Thematic Review Series: Lipid Posttranslational Modifications. Prelamin A, Zmpste24, misshapen cell nuclei, and progeria—new evidence suggesting that protein farnesylation could be important for disease pathogenesis

      Prelamin A undergoes multistep processing to yield lamin A, a structural protein of the nuclear lamina. Prelamin A terminates with a CAAX motif, which triggers farnesylation of a C-terminal cysteine (the C of the CAAX motif), endoproteolytic release of the last three amino acids (the AAX), and methylation of the newly exposed farnesylcysteine residue. In addition, prelamin A is cleaved a second time, releasing 15 more residues from the C terminus (including the farnesylcysteine methyl ester), generating mature lamin A. This second cleavage step is carried out by an endoplasmic reticulum membrane protease, ZMPSTE24. Interest in the posttranslational processing of prelamin A has increased with the recognition that certain progeroid syndromes can be caused by mutations that lead to an accumulation of farnesyl-prelamin A. Recently, we showed that a key cellular phenotype of these progeroid disorders, misshapen cell nuclei, can be ameliorated by inhibitors of protein farnesylation, suggesting a potential strategy for treating these diseases.
      In this article, we review the posttranslational processing of prelamin A, describe several mouse models for progeroid syndromes, explain the mutations underlying several human progeroid syndromes, and summarize recent data showing that misshapen nuclei can be ameliorated by treating cells with protein farnesyltransferase inhibitors.
      Many cellular proteins are enzymatically modified by the addition of a cholesterol biosynthetic intermediate, either a 15 carbon farnesyl or a 20 carbon geranylgeranyl lipid (
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ,
      • Clarke S.
      Protein isoprenylation and methylation at carboxyl-terminal cysteine residues.
      ,
      • Reiss Y.
      • Brown M.S.
      • Goldstein J.L.
      Divalent cation and prenyl pyrophosphate specificities of the protein farnesyltransferase from rat brain, a zinc metalloenzyme.
      ,
      • Seabra M.C.
      • Reiss Y.
      • Casey P.J.
      • Brown M.S.
      • Goldstein J.L.
      Protein farnesyltransferase and geranylgeranyltransferase share a common α subunit.
      ). This lipid modification, generally called protein prenylation, is important for facilitating the binding of proteins to membrane surfaces. Two broad classes of prenylated proteins exist. The Rab GTPases, important for trafficking of proteins to specific membrane compartments within cells, are geranylgeranylated at a pair of C-terminal cysteines (
      • Pereira-Leal J.B.
      • Seabra M.C.
      The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily.
      ,
      • Seabra M.C.
      • James G.L.
      Prenylation assays for small GTPases.
      ,
      • Seabra M.C.
      • Mules E.H.
      • Hume A.N.
      Rab GTPases, intracellular traffic and disease.
      ,
      • Brown M.S.
      • Goldstein J.L.
      Protein prenylation. Mad bet for Rab.
      ). A more abundant group of proteins terminate with a C-terminal CAAX motif and are either farnesylated or geranylgeranylated at a C-terminal cysteine (the C of the CAAX motif), then further modified by endoproteolytic processing and methylation of the C-terminal prenylcysteine (
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ,
      • Clarke S.
      Protein isoprenylation and methylation at carboxyl-terminal cysteine residues.
      ,
      • Young S.G.
      • Ambroziak P.
      • Kim E.
      • Clarke S.
      Postisoprenylation protein processing: CXXX (CaaX) endoproteases and isoprenylcysteine carboxyl methyltransferase.
      ,
      • Hrycyna C.A.
      • Clarke S.
      Modification of eukaryotic signaling proteins by C-terminal methylation reactions.
      ). Examples of prenylated CAAX proteins include regulatory proteins such as the Ras and Rho proteins, several nuclear lamins (prelamin A, lamin B1, and lamin B2), and the yeast mating pheromone a-factor.
      In this article, we focus largely on prelamin A, a farnesylated CAAX protein that undergoes multistep processing to yield mature lamin A, a structural protein of the nuclear lamina (
      • Lin F.
      • Worman H.J.
      Structural organization of the human gene encoding nuclear lamin A and nuclear lamin C.
      ,
      • Mounkes L.C.
      • Burke B.
      • Stewart C.L.
      The A-type lamins. Nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.
      ,
      • Burke B.
      • Stewart C.L.
      Life at the edge: the nuclear envelope and human disease.
      ,
      • Hutchison C.J.
      • Worman H.J.
      A-type lamins: guardians of the soma?.
      ). In addition to the aforementioned CAAX motif modifications, prelamin A undergoes an additional endoproteolytic cleavage reaction that produces mature lamin A; this cleavage step releases 15 amino acids from the C terminus of prelamin A, including the farnesylcysteine methyl ester. Hence, mature lamin A is not farnesylated.
      Interest in prelamin A and its posttranslational processing has recently increased with the recognition that certain progeroid syndromes can be caused by mutations that lead to an intracellular accumulation of farnesylated and carboxyl methylated prelamin A (
      • Eriksson M.
      • Brown W.T.
      • Gordon L.B.
      • Glynn M.W.
      • Singer J.
      • Scott L.
      • Erdos M.R.
      • Robbins C.M.
      • Moses T.Y.
      • Berglund P.
      • et al.
      Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome.
      ,
      • Fong L.G.
      • Ng J.K.
      • Meta M.
      • Cote N.
      • Yang S.H.
      • Stewart C.L.
      • Sullivan T.
      • Burghardt A.
      • Majumdar S.
      • Reue K.
      • et al.
      Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.
      ). Remarkably, at least one of the cellular phenotypes associated with these progeroid disorders, misshapen cell nuclei, can be reversed or ameliorated by inhibitors of protein farnesylation, suggesting a possible strategy for treating these disorders.
      In this article, we review the posttranslational processing of prelamin A, describe a mouse model for human progeroid syndromes, explain the mutations underlying several human progeroid syndromes, and summarize recent data indicating that the misshapen nuclei in certain progeroid syndromes can be ameliorated by treating cells with protein farnesyltransferase inhibitors (FTIs).
      All of the enzymes responsible for the processing of mammalian prelamin A were initially identified in Saccharomyces cerevisiae, an organism that does not express prelamin A. In yeast, these processing enzymes are essential for the biogenesis of a farnesylated mating pheromone, a-factor, from its precursor protein (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ,
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ,
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ,
      • Hrycyna C.A.
      • Sapperstein S.K.
      • Clarke S.
      • Michaelis S.
      The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and Ras proteins.
      ,
      • Hrycyna C.A.
      • Clarke S.
      Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae.
      ,
      • Powers S.
      • Michaelis S.
      • Broek D.
      • Santa Anna. S.
      • Field J.
      • Herskowitz I.
      • Wigler M.
      RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor.
      ,
      • He B.
      • Chen P.
      • Chen S-Y.
      • Vancura K.L.
      • Michaelis S.
      • Powers S.
      RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.
      ). Parallels between the posttranslational processing of the a-factor in yeast and prelamin A in mammals are both informative and intriguing. Therefore, we will begin this review by “backing up” to yeast and reviewing the biogenesis of yeast a-factor.

      POSTTRANSLATIONAL MODIFICATIONS OF CAAX PROTEINS

      CAAX proteins terminate with the amino acids CAAX, in which the C is a cysteine, the A residues are usually aliphatic amino acids, and the X can be one of many different residues (
      • Casey P.J.
      Biochemistry of protein prenylation.
      ,
      • Casey P.J.
      • Seabra M.C.
      Protein prenyltransferases.
      ). The CAAX sequence triggers three sequential enzymatic modifications (Fig. 1)(
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ,
      • Casey P.J.
      • Seabra M.C.
      Protein prenyltransferases.
      ). First, a 15 carbon farnesyl or a 20 carbon geranylgeranyl lipid is added to the thiol group of the cysteine by cytosolic protein prenyltransferases, either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ,
      • Casey P.J.
      • Seabra M.C.
      Protein prenyltransferases.
      ). In general, the cysteine is geranylgeranylated if the X is a leucine or phenylalanine; otherwise, it is farnesylated (
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ,
      • Schafer W.R.
      • Rine J.
      Protein prenylation: genes, enzymes, targets, and functions.
      ). Prelamin A terminates in CSIM and therefore is farnesylated; lamin B1 and lamin B2 are also farnesylated. Next, the last three amino acids (i.e., the AAX of the CAAX motif) are removed by a prenylprotein-specific endoprotease in the endoplasmic reticulum (ER) membrane (
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ). After endoproteolysis, the newly exposed isoprenylcysteine is methylated by an ER membrane prenylprotein-specific methyltransferase, isoprenylcysteine carboxyl methyltransferase (Icmt, designated Ste14p in yeast) (
      • Clarke S.
      • Vogel J.P.
      • Deschenes R.J.
      • Stock J.
      Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.
      ,
      • Dai Q.
      • Choy E.
      • Chiu V.
      • Romano J.
      • Slivka S.R.
      • Steitz S.A.
      • Michaelis S.
      • Philips M.R.
      Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum.
      ).
      Figure thumbnail gr1
      Fig. 1Posttranslational processing of proteins that terminate with a C-terminal CAAX motif. CAAX proteins terminate with the amino acids CAAX, in which the C is a cysteine, the A residues are usually aliphatic amino acids, and the X can be one of many different residues. CAAX proteins undergo three sequential enzymatic modifications. First, a 15 carbon farnesyl or a 20 carbon geranylgeranyl lipid is added to the thiol group of the cysteine by protein prenyltransferases, either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I. In general, the cysteine is geranylgeranylated if the X is a leucine or a phenylalanine; otherwise, it is farnesylated. Prelamin A terminates in CSIM; hence, it is farnesylated. Lamin B1, lamin B2, Ras proteins, and yeast a-factor are also farnesylated; the Rho and Rac proteins are geranylgeranylated.
      The CAAX motif modifications render the C terminus of the protein more hydrophobic, facilitating the binding of proteins to membrane surfaces (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Hancock J.F.
      • Magee A.I.
      • Childs J.E.
      • Marshall C.J.
      All ras proteins are polyisoprenylated but only some are palmitoylated.
      ). In addition, CAAX modifications have been shown to facilitate protein-protein interactions (shown both for yeast Ras and its effector and for a-factor and its receptor and transporter) (
      • Young S.G.
      • Ambroziak P.
      • Kim E.
      • Clarke S.
      Postisoprenylation protein processing: CXXX (CaaX) endoproteases and isoprenylcysteine carboxyl methyltransferase.
      ,
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Caldwell G.A.
      • Naider F.
      • Becker J.M.
      Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation.
      ,
      • Sapperstein S.
      • Berkower C.
      • Michaelis S.
      Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.
      ), enhance the metabolic stability of proteins (a-factor intermediates in yeast lacking FTase or Ste14p are degraded rapidly) (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Sapperstein S.
      • Berkower C.
      • Michaelis S.
      Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.
      ), and facilitate the targeting of proteins to specific sites within the cell (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ,
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Young S.G.
      Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
      ,
      • Bergo M.O.
      • Gavino B.J.
      • Hong C.
      • Beigneux A.P.
      • McMahon M.
      • Casey P.J.
      • Young S.G.
      Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf.
      ,
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ). For example, in yeast, Ras proteins that do not undergo endoproteolysis or carboxyl methylation are not properly localized to the plasma membrane (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Mislocalization of green fluorescent protein (GFP)-Ras2p in rce1Δ or ste14Δ yeast. The GFP-tagged Ras2p is at the plasma membrane in a wild-type strain but is mislocalized to internal membranes in the mutant rce1Δ and ste14Δ strains (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ) (W. K. Schmidt and S. Michaelis, unpublished data).

      PROCESSING OF MATING PHEROMONES IN S. CEREVISIAE

      The discovery of protein prenylation and carboxyl methylation arose from the structural analysis of the mating pheromones secreted by several relatively obscure heterobasidiomycetous jelly fungi (Tremella mesenterica, Tremella brasiliensis, and Rhodosporidium torluloides) (
      • Caldwell G.A.
      • Naider F.
      • Becker J.M.
      Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation.
      ,
      • Sakagami Y.
      • Yoshida M.
      • Isogai A.
      • Suzuki A.
      Peptidal sex hormones inducing conjugation tube formation in compatible mating-type cells of Tremella mesenterica.
      ,
      • Sakagami Y.
      • Isogai A.
      • Suzuki A.
      • Tamura S.
      • Kitada C.
      • Fujino M.
      Structure of tremerogen A-10, a peptidal hormone inducing conjugation tube formation in Tremella mesenterica.
      ,
      • Kamiya A.
      • Sakurai A.
      • Tamura S.
      • Takahashi N.
      • Tsuchiya E.
      • Abe K.
      • Fukui S.
      Structure of rhodotorucine A, a peptidyl factor, inducing mating tube formation in Rhodosporidium toruloides.
      ). The A-10 mating factor of T. mesenterica was found to contain an α-carboxyl methyl ester of a C-terminal cysteine residue, which was also modified by an S-farnesyl group (
      • Sakagami Y.
      • Yoshida M.
      • Isogai A.
      • Suzuki A.
      Peptidal sex hormones inducing conjugation tube formation in compatible mating-type cells of Tremella mesenterica.
      ,
      • Sakagami Y.
      • Isogai A.
      • Suzuki A.
      • Tamura S.
      • Kitada C.
      • Fujino M.
      Structure of tremerogen A-10, a peptidal hormone inducing conjugation tube formation in Tremella mesenterica.
      ). Similar findings were made on the A(Ia) mating factor of T. brasiliensis (
      • Ishibashi Y.
      • Sakagami Y.
      • Isogai A.
      • Suzuki A.
      Structures of tremerogens A-9291-I and A-9291-VIII: peptidyl sex hormones of Tremella brasiliensis.
      ,
      • Ishibashi Y.
      • Sakagami Y.
      • Isogai A.
      • Suzuki A.
      • Bandoni R.J.
      Isolation of tremerogens A-9291-I and A-9291-II, novel sex hormones of Tremella brasiliensis.
      ). All of the subsequent breakthroughs in understanding the CAAX processing machinery were made through the analysis of mating-defective mutants in S. cerevisiae. The mating process in S. cerevisiae (Fig. 3)involves two haploid cell types, MATa and MATα, which secrete the diffusible signaling molecules, a-factor and α-factor, respectively (
      • Sprague G.F.
      • Thorner J.W.
      Pheromone response and signal transduction during the mating process of Saccharomyces cerevisiae.
      ). The interaction of these mating pheromones with their cognate receptors leads to the formation of diploid yeast (Fig. 3). Despite the similarity in the function of a-factor and α-factor, the two pheromones are structurally and biochemically distinct. α-Factor is a nonlipidated peptide that is secreted by the classical secretory pathway, whereas a-factor is farnesylated and carboxyl methylated and is exported from yeast by a nonclassical secretion mechanism (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ,
      • Anderegg R.J.
      • Betz R.
      • Carr S.A.
      • Crabb J.W.
      • Duntze W.
      Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component.
      ,
      • Michaelis S.
      • Powers S.
      Biogenesis of yeast mating pheromone a-factor and RAS proteins.
      ,
      • Michaelis S.
      STE6, the yeast a-factor transporter.
      ).
      Figure thumbnail gr3
      Fig. 3Scheme of S. cerevisiae mating. The haploid yeast cell types (MATa and MATα) secrete the a-factor and α-factor pheromones, respectively, which initiate the mating process by interacting with receptors on the opposite cell type. The a-factor pheromone is a farnesylated and carboxyl methylated 12-mer, whereas α-factor is an unmodified 11-mer peptide (top) (
      • Sprague G.F.
      • Thorner J.W.
      Pheromone response and signal transduction during the mating process of Saccharomyces cerevisiae.
      ,
      • Anderegg R.J.
      • Betz R.
      • Carr S.A.
      • Crabb J.W.
      • Duntze W.
      Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component.
      ). The pheromone-receptor interaction stimulates a signaling pathway that leads to G1 cell cycle arrest and morphological alteration called shmoo formation (middle). Ultimately, the haploid cells fuse at the shmoo tips to form the MATa/α diploid zygote (bottom), and the cell cycle resumes (
      • Sprague G.F.
      • Thorner J.W.
      Pheromone response and signal transduction during the mating process of Saccharomyces cerevisiae.
      ).
      Studies from our laboratory (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ,
      • Hrycyna C.A.
      • Sapperstein S.K.
      • Clarke S.
      • Michaelis S.
      The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and Ras proteins.
      ,
      • Schmidt W.K.
      • Tam A.
      • Fujimura-Kamada K.
      • Michaelis S.
      Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ,
      • Schmidt W.K.
      • Tam A.
      • Michaelis S.
      Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis.
      ,
      • Tam A.
      • Schmidt W.K.
      • Michaelis S.
      The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.
      ) and others (
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ,
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ) have provided a detailed view of the a-factor biogenesis pathway (Fig. 4). Despite the small size of a-factor (the precursor and mature forms are 36 and 12 amino acids, respectively), the a-factor biogenesis pathway is complex, comprising three major events: C-terminal processing of the CAAX motif of the a-factor precursor (Fig. 4, steps 1–3), proteolytic removal of the N-terminal extension (steps 4 and 5), and secretion of mature bioactive a-factor (step 6) via an ATP binding cassette transporter, Ste6p.
      Figure thumbnail gr4
      Fig. 4Scheme of a-factor biogenesis. The a-factor precursor (P0) contains a C-terminal CAAX motif and an N-terminal extension and is encoded by the functionally redundant genes MFA1 (whose product is shown here) and MFA2. The CAAX motif directs an ordered series of C-terminal modifications (farnesylation, endoproteolytic cleavage, and carboxyl methylation; steps 1–3). The N-terminal extension undergoes two sequential proteolytic cleavages (steps 4 and 5). These modifications yield mature a-factor, a farnesylated and carboxyl methylated dodecapeptide (M), which is exported from the cell (step 6) by an ATP binding cassette transporter, Ste6p. The precursor (P0), the biosynthetic intermediates (P0*, P1, P2), and mature a-factor (M) can be distinguished by SDS-PAGE (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ,
      • Schmidt W.K.
      • Tam A.
      • Michaelis S.
      Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis.
      ,
      • Adames N.
      • Blundell K.
      • Ashby M.N.
      • Boone C.
      Role of yeast insulin-degrading enzyme homologs in propheromone processing and bud site selection.
      ). The gene products and corresponding enzymatic activities that mediate steps 1–6 of the a-factor biogenesis pathway are indicated. As described in the text, Ste24p has a role in two cleavage events associated with this pathway (steps 2 and 4). In its role as a CAAX protease, Ste24p performs a redundant function with Rce1p (step 2) to yield the P0* a-factor intermediate. As an N-terminal protease (step 4), Ste24p mediates the P1→P2 proteolytic cleavage. Both reactions can be carried out by purified Ste24p (
      • Tam A.
      • Schmidt W.K.
      • Michaelis S.
      The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.
      ).

      IDENTIFICATION OF ENZYMES RESPONSIBLE FOR THE C-TERMINAL PROCESSING OF a-FACTOR

      A major breakthrough in the CAAX processing field was the discovery of the RAM1 gene, which was identified in independent mutant screens as a suppressor of hyperactive Ras (supH) and as a mutant that was completely defective in mating and in the production of a-factor (ste16) (
      • Powers S.
      • Michaelis S.
      • Broek D.
      • Santa Anna. S.
      • Field J.
      • Herskowitz I.
      • Wigler M.
      RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor.
      ,
      • Michaelis S.
      • Powers S.
      Biogenesis of yeast mating pheromone a-factor and RAS proteins.
      ). The identification of RAM1 (named for Ras and a-factor maturation) in entirely different genetic screens suggested a commonality between Ras and a-factor (
      • Powers S.
      • Michaelis S.
      • Broek D.
      • Santa Anna. S.
      • Field J.
      • Herskowitz I.
      • Wigler M.
      RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor.
      ,
      • Fujiyama A.
      • Tsunasawa S.
      • Tamanoi F.
      • Sakiyama F.
      S-Farnesylation and methyl esterification of C-terminal domain of yeast RAS2 protein prior to fatty acid acylation.
      ). This commonality was underscored by the presence of a CAAX motif in both proteins. Ultimately, both Ras and a-factor were shown to be prenylated and methylated, and RAM1, along with a second gene, RAM2, was shown to encode different subunits of a heterodimeric enzyme, protein farnesyltransferase (
      • He B.
      • Chen P.
      • Chen S-Y.
      • Vancura K.L.
      • Michaelis S.
      • Powers S.
      RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.
      ).
      Ras and a-factor also require the same gene, STE14, for carboxyl methylation (Fig. 4, step 3). STE14 encodes an ICMT. This gene was initially identified by characterizing a sterile strain of yeast, ste14Δ, that lacked the capacity to produce a functional a-factor (
      • Hrycyna C.A.
      • Sapperstein S.K.
      • Clarke S.
      • Michaelis S.
      The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and Ras proteins.
      ,
      • Hrycyna C.A.
      • Clarke S.
      Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae.
      ,
      • Sapperstein S.
      • Berkower C.
      • Michaelis S.
      Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.
      ). Because Ste14p is an integral membrane protein of the ER membrane, the purification of the enzyme presented major challenges. However, heterologous expression studies (
      • Hrycyna C.A.
      • Sapperstein S.K.
      • Clarke S.
      • Michaelis S.
      The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and Ras proteins.
      ,
      • Hrycyna C.A.
      • Wait S.J.
      • Backlund Jr., P.S.
      • Michaelis S.
      Yeast STE14 methyltransferase, expressed as TrpE-STE14 fusion protein in Escherichia coli, for in vitro carboxylmethylation of prenylated polypeptides.
      ), and ultimately biochemical experiments with a purified Ste14p enzyme (
      • Anderson J.L.
      • Frase H.
      • Michaelis S.
      • Hrycyna C.A.
      Purification, functional reconstitution, and characterization of the Saccharomyces cerevisiae isoprenylcysteine carboxylmethyltransferase Ste14p.
      ), demonstrated that Ste14p is indeed an ICMT and that it is the sole enzyme for methylating CAAX proteins in yeast.
      Interestingly, the genes encoding CAAX endoprotease enzymes (Fig. 4, step 2) were never identified in standard genetic screens for sterile yeast defective in a-factor production. In hindsight, the failure to identify the endoproteases by simple genetic screens was entirely predictable, as we now know that two functionally redundant CAAX endoproteases exist in yeast, Ste24p and Rce1p, and both are capable of participating in the cleavage of the AAX from a-factor (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). Thus, a mutation in a single gene could not be expected to yield a sterile phenotype. The key to discovering RCE1 and AFC1 (another name for STE24) rested on experiments performed by Boyartchuk, Ashby, and Rine (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ) with an a-factor substrate that could be cleaved by Ste24p but not by Rce1p. They produced yeast expressing a mutant form of a-factor terminating in CAMQ rather than with the wild-type CVIA motif and demonstrated that the yeast remained capable of producing mature a-factor. The mutant yeast expressing the CAMQ version of a-factor were then mutagenized, and an autocrine arrest selection strategy was used to isolate sterile mutants (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). This approach resulted in the identification of a sterile yeast mutant, and the responsible gene, STE24, was cloned by complementation. STE24 encodes a 453 amino acid protein, Ste24p, with multiple predicted transmembrane helices as well as an HEXXH (H, His; E, Glu) motif characteristic of a group of zinc-dependent metalloproteases (
      • Schmidt W.K.
      • Michaelis S.
      Ste24 Protease.
      ). Mutating either of the conserved histidines in the HEXXH domain blocked the ability of Ste24p to complement the mating defect of STE24-deficient yeast (ste24Δ) expressing the CAMQ form of a-factor (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ).
      STE24-deficient yeast expressing wild-type a-factor secreted reduced amounts of a-factor but were not sterile, suggesting the existence of another gene capable of processing a-factor. The residual CAAX endoprotease activity in ste24Δ yeast membranes was insensitive to the zinc chelator 1,10-o-phenanthroline, suggesting that the remaining endoprotease was not a zinc metalloprotease (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). To isolate the remaining endoprotease, ste24Δ yeast were mutagenized and screened for a mutation that blocked residual a-factor production. This approach led to the identification of RCE1, which encodes a 329 amino acid protein that is also predicted to have multiple transmembrane helices (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). Unlike Ste24p, Rce1p does not contain sequences characteristic of any of the defined classes of proteases, but there were remote similarities with the type IIb signal peptidase (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). Yeast lacking RCE1 (rce1Δ) manifest a modest decrease in a-factor production, and membranes from rce1Δ yeast had moderately decreased CAAX endoprotease activity in in vitro assays with farnesylated a-factor peptide substrates. The residual CAAX endoprotease activity in rce1Δ yeast was sensitive to o-phenanthroline, consistent with Ste24p being a zinc metalloprotease (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ).
      The functional redundancy of Ste24p and Rce1p for processing a-factor with a wild-type CAAX motif can be readily demonstrated with a yeast halo assay (Fig. 5)(
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ,
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ,
      • Trueblood C.E.
      • Boyartchuk V.L.
      • Picologlou E.A.
      • Rozema D.
      • Poulter C.D.
      • Rine J.
      The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.
      ,
      • Michaelis S.
      • Herskowitz I.
      The a-factor pheromone of Saccharomyces cerevisiae is essential for mating.
      ). In this assay, the production of mature a-factor can be visualized by a zone of growth inhibition (halo) in a lawn of yeast with an α-mating type. The single mutants (ste24Δ and rce1Δ) have modestly reduced a-factor production and therefore yield slightly smaller halos, whereas the double mutant (ste24Δrce1Δ) fails to carry out CAAX endoproteolysis, resulting in the complete inability to produce mature a-factor and the complete absence of a halo (Fig. 5).
      Figure thumbnail gr5
      Fig. 5The CAAX endoproteases Ste24p and Rce1p exhibit distinct, but overlapping, substrate specificities. Plasmids encoding the yeast a-factor gene (MFA1) with its normal CAAX motif (CVIA; row 3) or CAAX variants (rows 1, 2, and 4) were transformed into the indicated yeast strains that lack none [wild type (wt)], one (ste24Δ or rce1Δ), or both (ste24Δrce1Δ) of the CAAX endoprotease genes. The relative levels of a-factor produced by these strains were evaluated by the a-factor “halo assay.” In this assay, MATa cells are spotted onto a lawn of MATα sst2 cells that are hypersensitive to a-factor. The zone of growth inhibition (halo) reflects the amount of a-factor produced by the cells that are spotted. Only properly processed a-factor results in a halo. The variants tested here, from top to bottom, are CTLM from Ste18p, which like the Ras2p CAAX motif (CIIS) is Rce1p-specific (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ,
      • Trueblood C.E.
      • Boyartchuk V.L.
      • Picologlou E.A.
      • Rozema D.
      • Poulter C.D.
      • Rine J.
      The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.
      ); CAMQ from the mammalian phosphorylase kinase α subunit, which is Ste24p-specific (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ); CVIA, the a-factor CAAX motif, which can be cleaved by either Rce1p or Ste24p (
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ,
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ); and CSIM, the prelamin CAAX motif, which appears to be Rce1p-specific (M. Boyle and S. Michaelis, unpublished data).
      Ste24p and Rce1p have different abilities to cleave a-factor mutants with different CAAX motifs. An a-factor mutant with the CAMQ sequence motif can only be cleaved by Ste24p, whereas other a-factor mutants, such as one terminating in CTLM, are cleaved solely by Rce1p (Fig. 5) (
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ). Notably, when the CAAX motif for mammalian prelamin A, CSIM, is tested in the a-factor assay, it is cleaved mainly by Rce1p (Fig. 5). The sequence specificities of Ste24p and Rce1p in the context of a-factor have been examined by Rine and colleagues (
      • Trueblood C.E.
      • Boyartchuk V.L.
      • Picologlou E.A.
      • Rozema D.
      • Poulter C.D.
      • Rine J.
      The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.
      ). However, a complete picture of the specificities of these two enzymes has not yet emerged, either for a-factor or for any other CAAX protein, and it is not clear whether any additional structural features, aside from the sequence of the CAAX motif, influence enzyme specificity.

      A SECOND ROLE FOR STE24 IN THE BIOGENESIS OF a-FACTOR

      A surprising discovery, and one quite relevant to this review, was our finding that one of the a-factor-processing enzymes, Ste24p, actually plays dual roles in a-factor maturation. As shown in Fig. 4, Ste24p mediates both the C-terminal CAAX endoproteolysis (step 2) and the first N-terminal cleavage (step 4) (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ,
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ,
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ). In addition to the CAAX motif modifications on the C terminus of a-factor (farnesylation, endoproteolytic release of the AAX, and methylation), two additional endoprotease steps within the N-terminal portion of the molecule are required to generate mature a-factor. The first of these processing steps, which converts the P1 intermediate to P2, involves the endoproteolytic release of the first seven amino acids of the protein (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ). This cleavage reaction was shown by Edman sequencing to occur between threonine-7 and alanine-8 and is blocked in an Ala8Gly a-factor mutant (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ). The second cleavage reaction, which coverts the P2 intermediate to mature a-factor, involves the release of an additional 14 amino acids (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Anderegg R.J.
      • Betz R.
      • Carr S.A.
      • Crabb J.W.
      • Duntze W.
      Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component.
      ).
      Metabolic labeling experiments of wild-type yeast have shown that P1 is initially converted to P2, after which the P2 intermediate is further processed to mature a-factor (Fig. 4, steps 4 and 5) (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ). Genetic experiments have shown that Axl1p plays a key role in the second of the two N-terminal processing steps, converting P2 to mature a-factor (
      • Adames N.
      • Blundell K.
      • Ashby M.N.
      • Boone C.
      Role of yeast insulin-degrading enzyme homologs in propheromone processing and bud site selection.
      ). In axl1Δ yeast, the P2 intermediate of a-factor accumulates. A search of the S. cerevisiae sequence database revealed a gene similar to AXL1, designated STE23, and this gene product can also participate in the second N-terminal proteolysis step. Yeast lacking AXL1 have a reduced capacity to produce a-factor, whereas mutants lacking both AXL1 and STE23 (axl1Δste23Δ) are completely sterile (
      • Adames N.
      • Blundell K.
      • Ashby M.N.
      • Boone C.
      Role of yeast insulin-degrading enzyme homologs in propheromone processing and bud site selection.
      ).
      Our studies revealed that the first of the two N-terminal cleavage reactions, the one that converts P1 to P2, is dependent on STE24 and can be blocked by mutating the HEXXH motif in Ste24p (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ). In the absence of STE24, the P1 intermediate accumulates (Fig. 6). Interestingly, this N-terminal cleavage reaction was the first role to be ascribed to Ste24p (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ), followed soon thereafter by the report demonstrating the role of Ste24p in the release of the AAX (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). The apparent conflict posed by reports indicating different roles in a-factor biogenesis was quickly resolved by additional genetic and biochemical studies, which showed that Ste24p actually has dual roles in a-factor processing. Along with Rce1p, Ste24p is capable of releasing the AAX from a-factor; Ste24p also clearly cleaves the first seven amino acids from the protein (
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ). Yeast lacking STE24 exhibit a strikingly reduced capacity to produce mature a-factor; only a small amount of mature a-factor, perhaps 5% of the normal amount, is still made (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ,
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ). The most likely explanation for the residual a-factor production is that some Axl1p/Ste23p-mediated cleavage of a-factor occurs in the absence of Ste24p, albeit at reduced efficiency.
      Figure thumbnail gr6
      Fig. 6The P1 intermediate of a-factor accumulates in ste24Δ yeast. The N-terminal (P1→P2) processing of a-factor is defective in a ste24Δ mutant. Processing of the a-factor precursor in wild-type (WT) and ste24Δ mutant cells is shown. Cells were pulse-labeled with [35S]cysteine for 5 min and chased for 30 min. The intracellular fraction was subjected to immunoprecipitation with a-factor antibodies, electrophoresis, and autoradiography. The P1, P2, and M forms of a-factor are indicated. P1 accumulates in the ste24Δ mutant. This figure is adopted from The Journal of Cell Biology (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ), and reproduced by copyright permission of Rockefeller University Press.
      Neither the C- nor the N-terminal cleavage of a-factor occurs in the absence of farnesylation, as shown by the complete lack of proteolytic intermediates in ram1Δ or ram2Δ mutants (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • He B.
      • Chen P.
      • Chen S-Y.
      • Vancura K.L.
      • Michaelis S.
      • Powers S.
      RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.
      ). Whether farnesylation is required for substrate recognition and specificity, or simply facilitates the recruitment of the a-factor intermediate to membranes containing Ste24p, has not yet been determined. However, the N-terminal cleavage reaction mediated by Ste24p does not require prior carboxyl methylation, because N-terminal cleavage occurs normally in yeast lacking STE14 (
      • Sapperstein S.
      • Berkower C.
      • Michaelis S.
      Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.
      ). The precise sequences and structures within the a-factor required for the two N-terminal cleavage reactions have not yet been determined, but with the experimental systems that have been developed, it should be possible to obtain that type of information.
      Recently, a histidine-tagged version of Ste24p was detergent-solubilized and purified to homogeneity from yeast and shown to be the sole protein needed to mediate both the C- and N-terminal a-factor cleavage reactions (
      • Tam A.
      • Schmidt W.K.
      • Michaelis S.
      The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.
      ). Given that fact that Ste24p is a membrane protein, the retention of enzymatic activities after purification was remarkable. Both of the activities were blocked by o-phenanthroline and reactivated by zinc (
      • Tam A.
      • Schmidt W.K.
      • Michaelis S.
      The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.
      ).

      THE STE24 ORTHOLOG IN MAMMALS, ZMPSTE24, COMPLEMENTS STE24 DEFICIENCY IN YEAST

      The genes encoding the mammalian postisoprenylation enzymes (ZMPSTE24, RCE1, and ICMT) were identified by homology with their yeast counterparts (STE24, RCE1, and STE14, respectively) (
      • Dai Q.
      • Choy E.
      • Chiu V.
      • Romano J.
      • Slivka S.R.
      • Steitz S.A.
      • Michaelis S.
      • Philips M.R.
      Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ,
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      Cloning and characterization of a mammalian prenyl protein-specific protease.
      ). In each case, the mammalian gene complements the yeast mutant, as judged by a conventional a-factor halo assay and yeast mating tests. In this regard, our studies of human ZMPSTE24 deserve special mention because we were able to demonstrate its ability to complement both the C- and N-terminal endoproteolytic processing reactions of a-factor (
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ). This point is illustrated in Fig. 7, in which complementation of ste24Δrce1Δ yeast with human ZMPSTE24 is shown by three separate assays (metabolic labeling, halo, and mating). Mouse Zmpste24 also fully complements the a-factor production defect in ste24Δrce1Δ yeast (see Fig. 13A below) (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ). The complementation of the mutant yeast was fascinating, particularly because signaling molecules resembling a-factor have never been identified in mammals. Thus, mammalian Zmpste24 retains the capacity to process a yeast substrate after nearly 800 million years of evolution. The ability of the mammalian enzyme to process yeast a-factor has facilitated the functional analysis of mutant forms of ZMPSTE24 in humans (
      • Agarwal A.K.
      • Fryns J-P.
      • Auchus R.J.
      • Garg A.
      Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia.
      ).
      Figure thumbnail gr7
      Fig. 7The human STE24 homolog, ZMPSTE24, complements the a-factor biosynthesis, halo, and mating defects when expressed in ste24Δrce1Δ mutant yeast. The human ZMPSTE24 gene was tested in parallel with yeast STE24 for its ability to complement the a-factor defect of a double (ste24Δrce1Δ) mutant strain. Using this mutant, the yeast or human protein must carry out both the C-terminal CAAX processing and N-terminal processing of a-factor to generate mature bioactive a-factor. Cells transformed with empty vector, yeast STE24, or human ZMPSTE24 were tested for their ability to produce the P1, P2, and M (mature) a-factor in a metabolic labeling experiment (A) and in a-factor halo and mating assays (B) (
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ,
      • Tam A.
      • Schmidt W.K.
      • Michaelis S.
      The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.
      ). In the mating test, patches of auxotrophic MATa cells are replica-plated to a lawn of MATα cells with complementary auxotrophy. Where mating occurs, prototrophic diploids are formed that can grow in medium with no supplements. I, intracellular; E, extracellular. This figure is adapted from figures published in The Journal of Cell Biology (
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ) and The Journal of Biological Chemistry (
      • Schmidt W.K.
      • Tam A.
      • Michaelis S.
      Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis.
      ) and is reproduced with permission.
      Figure thumbnail gr13
      Fig. 13A: Yeast halo assay demonstrating that the expression of yeast RCE1, mouse Rce1, yeast STE24, and mouse Zmpste24 restores the production of a-factor in ste24Δrce1Δ yeast. A mutant mouse Zmpste24 construct lacking exon 8 sequences (which encode the HEXXH motif) did not restore a-factor production. B: Reduced ability of membranes from Zmpste24−/− tissues to carry out the N-terminal processing of a-factor. A 35S-labeled P1 a-factor intermediate was prepared from a yeast strain that lacked STE24 and expressed MFA1 from a high-copy plasmid. Proteolysis reactions were performed by incubating the radiolabeled P1 with mouse membranes for 2 h at 30°C in the absence or presence of 1,10-phenanthroline. The a-factor intermediates were then immunoprecipitated with an a-factor-specific antiserum and size-fractionated by SDS-PAGE. Dried gels were analyzed by autoradiography and with a phosphor imager. The N-terminal processing reaction cleaves seven amino acids from the N terminus of P1, yielding the shorter intermediate. Reproduced, with permission, from The Journal of Biological Chemistry (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ).
      It is worth pointing out that multiple names have been used in the literature for yeast STE24 and mammalian Zmpste24. First cloned and named STE24 in yeast (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ), the gene was later called AFC1 (for a-factor-converting enzyme) (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). Currently, the STE24 designation is used exclusively by bioinformatics databases and the scientific community. Different names have also been applied to the mammalian gene Zmpste24. We cloned the first full-length mammalian version of Zmpste24 and initially designated it HsSTE24 (for Homo sapiens STE24) (
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ). Another group called it HsSte24 (
      • Kumagai H.
      • Kawamura Y.
      • Yanagisawa K.
      • Komano H.
      Identification of a human cDNA encoding a novel protein structurally related to the yeast membrane-associated metalloprotease, Ste24p.
      ). The official name for the human and mouse genes has since been changed to Zmpste24 (to reflect the fact that this enzyme is an ortholog of Ste24p and is a zinc metalloprotease). One group came up with entirely new names for Ste24p/Zmpste24 and Rce1, calling them FACE-1 and FACE-2, for farnesylated protein-converting enzymes 1 and 2, respectively (
      • Freije J.M.P.
      • Blay P.
      • Pendás A.M.
      • Cadiñanos J.
      • Crespo P.
      • López-Otín C.
      Identification and chromosomal location of two human genes encoding enzymes potentially involved in proteolytic maturation of farnesylated proteins.
      ). Those names were less than ideal choices, in our opinion, because it was well established at the time that at least one of the endoprotease activities, now recognized to be Rce1, processes both farnesylated and geranylgeranylated proteins, not just farnesylated proteins.

      PHYSIOLOGIC IMPORTANCE OF RCE1 AND ICMT IN MAMMALS AND MICE

      Deciphering the physiologic importance of CAAX processing in mammals has been greatly aided by the development of Rce1 and Icmt knockout mice (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Gomes A.Q.
      • Seabra M.C.
      • Young S.G.
      Isoprenylcysteine carboxyl methyltransferase deficiency in mice.
      ). Nearly all homozygous knockout mice (Rce1−/−) died fairly late in gestation, beginning at embryonic day 14.5–15.5 (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ). Very rarely, Rce1−/− mice were born alive, but they were runted and lived for only a few weeks. Rce1 deficiency had no significant effect on hematopoiesis, and the major organ systems of Rce1−/− embryos were histologically normal late in gestation (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ).
      Rce1 is absolutely essential for the endoproteolytic processing of the Ras proteins in mice. The electrophoretic mobility of Ras proteins is distinctly abnormal in Rce1−/− embryo lysates (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ). Because endoproteolysis is a prerequisite for carboxyl methylation, we predicted that Ras proteins from Rce1−/− fibroblasts would not be methylated. Indeed, this was the case. When we labeled Rce1+/+ and Rce1−/− fibroblasts with l-[methyl-3H]methionine, the Ras proteins from Rce1+/+ fibroblasts contained a 3H-labeled farnesylcysteine methyl ester, whereas the Ras proteins from Rce1−/− fibroblasts did not (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ).
      Crude membrane fractions from Rce1+/+ fibroblasts were capable of cleaving the AAX from recombinant H-Ras, N-Ras, and K-Ras, regardless of whether they were farnesylated or geranylgeranylated (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      Cloning and characterization of a mammalian prenyl protein-specific protease.
      ). In contrast, membranes from Rce1−/− fibroblasts did not process any of the Ras proteins. Thus, as in yeast (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ), the endoproteolytic processing of the Ras proteins requires Rce1. The endoproteolytic processing of other CAAX proteins was also blocked (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      Cloning and characterization of a mammalian prenyl protein-specific protease.
      ). Interestingly, Rce1 is required for the endoproteolytic processing of lamin B1. Maske et al. (
      • Maske C.P.
      • Hollinshead M.S.
      • Higbee N.C.
      • Bergo M.O.
      • Young S.G.
      • Vaux D.J.
      A carboxyl-terminal interaction of lamin B1 is dependent on the CAAX endoprotease Rce1 and carboxymethylation.
      ) generated a monoclonal antibody specific for the C terminus of lamin B1, but the antibody did not bind unless the protein had undergone both farnesylation and endoproteolytic processing. Of note, the monoclonal antibody did not detect lamin B1 in Rce1−/− fibroblasts, indicating that Rce1 is solely responsible for the processing of that protein. Because Rce1 has so many protein substrates, it was not surprising that biochemical studies revealed a substantial accumulation of uncleaved and unmethylated CAAX protein substrates in Rce1−/− fibroblasts (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ).
      In Rce1+/+ fibroblasts, the Ras proteins are located along the inner surface of the plasma membrane, as judged by cell transfection experiments with green fluorescent protein (GFP)-Ras fusion constructs (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ). In contrast, the Ras proteins in Rce1−/− fibroblasts were strikingly mislocalized to the cytosol and internal membrane compartments (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ), similar to what is seen in the yeast mutant (Fig. 2), underscoring the importance of endoproteolysis (and subsequent methylation) for proper membrane targeting.
      The Rce1 knockout had functionally important consequences. Rce1−/− fibroblasts grew slightly more slowly than Rce1+/+ fibroblasts and were less susceptible to oncogenic transformation by activated forms of Ras (
      • Bergo M.O.
      • Ambroziak P.
      • Gregory C.
      • George A.
      • Otto J.C.
      • Kim E.
      • Nagase H.
      • Casey P.J.
      • Balmain A.
      • Young S.G.
      Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation.
      ). Also, we generated mice homozygous for a conditional Rce1 allele and then showed that Cre-mediated inactivation of Rce1 in fibroblasts reduces transformation by mutationally activated forms of Ras (
      • Bergo M.O.
      • Ambroziak P.
      • Gregory C.
      • George A.
      • Otto J.C.
      • Kim E.
      • Nagase H.
      • Casey P.J.
      • Balmain A.
      • Young S.G.
      Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation.
      ).
      We also created mice lacking Icmt (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Young S.G.
      Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
      ) and to our surprise found that this gene defect caused a more severe phenotype than Rce1 deficiency. Icmt−/− embryos were viable until E10.5, but they all died by E12.5. One study contended that Icmt might be particularly important for the development of the liver (
      • Lin X.
      • Jung J.
      • Kang D.
      • Xu B.
      • Zaret K.S.
      • Zoghbi H.
      Prenylcysteine carboxylmethyltransferase is essential for the earliest stages of liver development in mice.
      ). Icmt also might be important for the development of the brain; Icmt−/− embryonic stem (ES) cells completely lacked the ability to contribute to the formation of the brain (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Gomes A.Q.
      • Seabra M.C.
      • Young S.G.
      Isoprenylcysteine carboxyl methyltransferase deficiency in mice.
      ).
      We were successful in culturing Icmt−/− fibroblasts from E11.5 mouse embryos (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Gomes A.Q.
      • Seabra M.C.
      • Young S.G.
      Isoprenylcysteine carboxyl methyltransferase deficiency in mice.
      ). Icmt−/− fibroblasts lacked methyltransferase activity against both small-molecule Icmt substrates (e.g., N-acetyl-farnesylcysteine) and farnesylated proteins such as K-Ras (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Young S.G.
      Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
      ). Icmt deficiency led to a substantial accumulation of unmethylated Icmt protein substrates within cells (i.e., proteins susceptible to methylation in the presence of recombinant Icmt and the methyl donor S-adenosylmethionine) (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Young S.G.
      Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
      ). Icmt is the only enzyme for methylating prenylcysteines in mammalian cells, methylating both CAAX proteins and the CXC subset (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Gomes A.Q.
      • Seabra M.C.
      • Young S.G.
      Isoprenylcysteine carboxyl methyltransferase deficiency in mice.
      ,
      • Smeland T.E.
      • Seabra M.C.
      • Goldstein J.L.
      • Brown M.S.
      Geranylgeranylated Rab proteins terminating in Cys-Ala-Cys, but not Cys-Cys, are carboxyl-methylated by bovine brain membranes in vitro.
      ) of geranylgeranylated Rab proteins. Primary Icmt−/− fibroblasts clearly grow more slowly than wild-type fibroblasts.
      The Ras proteins in Icmt−/− fibroblasts were not methylated, resulting in a very subtle retardation in electrophoretic mobility on SDS-polyacrylamide gels. Also, the Ras proteins were mislocalized in Icmt−/− fibroblasts, as judged by transfection experiments with GFP–Ras fusion constructs (Fig. 8)(
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ,
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Gomes A.Q.
      • Seabra M.C.
      • Young S.G.
      Isoprenylcysteine carboxyl methyltransferase deficiency in mice.
      ). Recent studies have shown that methylation is important for CAAX protein targeting within cells when the protein is farnesylated (
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ). When the CAAX protein is geranylgeranylated, the contribution of methylation to intracellular targeting appears to be negligible or nonexistent (
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ,
      • Silvius J.R.
      • l'Heureux F.
      Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers.
      ).
      Figure thumbnail gr8
      Fig. 8Mislocalization of K-Ras in spontaneously immortalized Icmt−/− fibroblasts that had been transfected with a GFP-K-Ras fusion construct (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Young S.G.
      Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
      ,
      • Bergo M.O.
      • Gavino B.J.
      • Hong C.
      • Beigneux A.P.
      • McMahon M.
      • Casey P.J.
      • Young S.G.
      Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf.
      ). The Ras proteins are similarly mislocalized in Rce1−/− fibroblasts (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ). Several of these images have been published previously in The Journal of Clinical Investigation (
      • Bergo M.O.
      • Gavino B.J.
      • Hong C.
      • Beigneux A.P.
      • McMahon M.
      • Casey P.J.
      • Young S.G.
      Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf.
      ) and The Enzymes (
      • Young S.G.
      • Clarke S.
      • Bergo M.
      • Philips M.
      • Fong L.G.
      Genetic approaches to understanding the physiologic importance of the carboxyl methylation of isoprenylated proteins.
      ) and are reproduced with permission.
      We produced mice that are homozygous for a conditional Icmt allele (Icmt fl/fl). Cre-mediated inactivation of Icmt mislocalized Ras proteins within cells, changed the steady-state concentration of both Rho and Ras proteins, and reduced the ability of mutationally activated Ras proteins to transform cultured fibroblasts (
      • Bergo M.O.
      • Gavino B.J.
      • Hong C.
      • Beigneux A.P.
      • McMahon M.
      • Casey P.J.
      • Young S.G.
      Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf.
      ). The latter finding has prompted interest in Icmt inhibitors as possible anticancer agents (
      • Anderson J.L.
      • Henriksen B.S.
      • Gibbs R.A.
      • Hrycyna C.A.
      The isoprenoid substrate specificity of isoprenylcysteine carboxylmethyltransferase: development of novel inhibitors.
      ,
      • Winter-Vann A.M.
      • Casey P.J.
      Post-prenylation-processing enzymes as new targets in oncogenesis.
      ,
      • Winter-Vann A.M.
      • Baron R.A.
      • Wong W.
      • Cruz J. dela
      • York J.D.
      • Gooden D.M.
      • Bergo M.O.
      • Young S.G.
      • Toone E.J.
      • Casey P.J.
      A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells.
      ).
      Previous pharmacologic studies with inhibitors of protein farnesyltransferase had shown that farnesylation is critical for the proper intracellular targeting of CAAX proteins (
      • Kohl N.E.
      • Wilson F.R.
      • Mosser S.D.
      • Giuliani E.
      • DeSolms S.J.
      • Conner M.W.
      • Anthony N.J.
      • Holtz W.J.
      • Gomez R.P.
      • Lee T-J.
      • et al.
      Protein farnesyltransferase inhibitors block the growth of ras-dependent tumors in nude mice.
      ,
      • Gibbs J.B.
      • Oliff A.
      • Kohl N.E.
      Farnesyltransferase inhibitors: Ras research yields a potential cancer therapeutic.
      ,
      • Tamanoi F.
      Inhibitors of Ras farnesyltransferases.
      ,
      • James G.L.
      • Goldstein J.L.
      • Brown M.S.
      • Rawson T.E.
      • Somers T.C.
      • McDowell R.S.
      • Crowley C.W.
      • Lucas B.K.
      • Levinson A.D.
      • C. Marsters Jr, J.
      Benzodiazepine peptidomimetics: potent inhibitors of Ras farnesylation in animal cells.
      ). Studies with Rce1- and Icmt-deficient fibroblasts (
      • Young S.G.
      • Ambroziak P.
      • Kim E.
      • Clarke S.
      Postisoprenylation protein processing: CXXX (CaaX) endoproteases and isoprenylcysteine carboxyl methyltransferase.
      ,
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Young S.G.
      Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
      ,
      • Bergo M.O.
      • Gavino B.J.
      • Hong C.
      • Beigneux A.P.
      • McMahon M.
      • Casey P.J.
      • Young S.G.
      Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf.
      ,
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ,
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Gomes A.Q.
      • Seabra M.C.
      • Young S.G.
      Isoprenylcysteine carboxyl methyltransferase deficiency in mice.
      ) have shown that the endoprotease and methyltransferase steps are also important for the proper targeting of some CAAX proteins in cells. We believe that these findings suggest a valuable lesson: whenever CAAX proteins are implicated in human disease, it is important to consider whether the disease might conceivably be treated by “attacking” the posttranslational modifications, thereby interfering with the localization and function of these proteins within cells.

      NUCLEAR LAMINS: ABUNDANT MAMMALIAN CAAX PROTEINS

      Lamin A, lamin C, lamin B1, and lamin B2 are intermediate filament proteins and are among the most abundant CAAX proteins in mammalian cells (
      • Zhang F.L.
      • Casey P.J.
      Protein prenylation: molecular mechanisms and functional consequences.
      ,
      • Schmidt R.A.
      • Schneider C.J.
      • Glomset J.A.
      Evidence for post-translational incorporation of a product of mevalonic acid into Swiss 3T3 cell proteins.
      ,
      • Glomset J.A.
      • Farnsworth C.C.
      Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes.
      ,
      • Kim C.M.
      • Goldstein J.L.
      • Brown M.S.
      cDNA cloning of MEV, a mutant protein that facilitates cellular uptake of mevalonate, and identification of the point mutation responsible for its gain of function.
      ). These proteins are key structural components of the nuclear lamina, a filamentous meshwork that lies beneath the inner nuclear membrane. Each of the lamin proteins contains an N-terminal globular domain, a central helical rod domain, and a C-terminal globular domain (
      • Mounkes L.C.
      • Burke B.
      • Stewart C.L.
      The A-type lamins. Nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.
      ,
      • Hutchison C.J.
      • Worman H.J.
      A-type lamins: guardians of the soma?.
      ). The lamin monomers dimerize to form parallel coiled-coil homodimers, which then associate head-to-tail to form strings and ultimately higher order 10 nm thick filaments that form the meshwork. It is unclear whether the different lamins form heterodimers. The nuclear lamins interact with a variety of integral membrane proteins of the inner nuclear membrane and also with transcription factors, as well as with heterochromatin itself (
      • Mounkes L.C.
      • Burke B.
      • Stewart C.L.
      The A-type lamins. Nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.
      ,
      • Wilson K.L.
      The nuclear envelope, muscular dystrophy and gene expression.
      ). Thus, these proteins have many roles besides simply forming a passive scaffold for the nuclear envelope.
      Prelamin A, lamin B1, and lamin B2, but not lamin C, contain CAAX motifs and undergo the usual CAAX motif modifications (isoprenylation, endoproteolysis, and methylation) (Fig. 9). In the case of prelamin A, the release of the AAX is likely a redundant function of Zmpste24 and Rce1 (
      • Bergo M.O.
      • Gavino B.
      • Ross J.
      • Schmidt W.K.
      • Hong C.
      • Kendall L.V.
      • Mohr A.
      • Meta M.
      • Genant H.
      • Jiang Y.
      • et al.
      Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.
      ,
      • Bergo M.O.
      • Young S.G.
      Zmpste24 (mammalian farnesylated protein-converting enzyme 1).
      ,
      • Corrigan D.P.
      • Kuszczak D.
      • Rusinol A.E.
      • Thewke D.P.
      • Hrycyna C.A.
      • Michaelis S.
      • Sinensky M.S.
      Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24.
      ). As noted previously, however, prelamin A undergoes a second endoproteolytic processing step (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ). The last 15 amino acids of the protein (including the farnesylcysteine methyl ester) are clipped off and degraded, leaving behind mature lamin A (Fig. 9). This second proteolytic processing step is carried out by Zmpste24 (
      • Corrigan D.P.
      • Kuszczak D.
      • Rusinol A.E.
      • Thewke D.P.
      • Hrycyna C.A.
      • Michaelis S.
      • Sinensky M.S.
      Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24.
      ). In the absence of that enzyme, farnesyl-prelamin A accumulates within cells (Fig. 10), specifically at the nuclear envelope (Fig. 11)(
      • Fong L.G.
      • Ng J.K.
      • Meta M.
      • Cote N.
      • Yang S.H.
      • Stewart C.L.
      • Sullivan T.
      • Burghardt A.
      • Majumdar S.
      • Reue K.
      • et al.
      Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.
      ,
      • Pendás A.M.
      • Zhou Z.
      • Cadiñanos J.
      • Freije J.M.P.
      • Wang J.
      • Hultenby K.
      • Astudillo A.
      • Wernerson A.
      • Rodríguez F.
      • Tryggvason K.
      • et al.
      Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice.
      ).
      Figure thumbnail gr9
      Fig. 9Biogenesis of lamin A in normal cells and the failure to generate mature lamin A in the setting of Zmpste24 deficiency. Left: Formation of lamin A from prelamin A in wild-type cells. Prelamin A (664 amino acids) undergoes four posttranslational processing steps. First, the cysteine of the CAAX motif is farnesylated by FTase. Second, the AAX is released. Third, the newly exposed farnesylcysteine is methylated. Fourth, the C-terminal 15 amino acids, including the farnesylcysteine methyl ester, are clipped off (by Zmpste24) and then degraded, leaving mature lamin A (646 amino acids). Right: Defective prelamin A processing in the setting of Zmpste24 deficiency. By analogy to a-factor biogenesis in yeast (
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
      ,
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ,
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ), we suspect that the AAX is probably released by Rce1 in Zmpste24−/− cells. Another reason to suspect that Rce1 could release the AAX is the observation that Rce1 cleaves the AAX from lamin B1 (
      • Maske C.P.
      • Hollinshead M.S.
      • Higbee N.C.
      • Bergo M.O.
      • Young S.G.
      • Vaux D.J.
      A carboxyl-terminal interaction of lamin B1 is dependent on the CAAX endoprotease Rce1 and carboxymethylation.
      ). Blocking farnesylation with a farnesyltransferase inhibitor (FTI) would mean that the C terminus of progerin would terminate with an α-carboxylate anion rather than a farnesylcysteine methyl ester; this change would reduce the hydrophobicity of the C terminus of the protein (
      • Silvius J.R.
      • l'Heureux F.
      Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers.
      ) and would be expected to reduce the avidity of the molecule for the inner nuclear membrane and possibly influence its interactions with other nuclear proteins.
      Figure thumbnail gr10
      Fig. 10Western blots of extracts from wild-type and Zmpste24−/− fibroblasts with a C-terminal prelamin A antibody and an N-terminal lamin A/C antibody. Reproduced, with permission, from The Proceedings of the National Academy of Sciences USA (
      • Fong L.G.
      • Ng J.K.
      • Meta M.
      • Cote N.
      • Yang S.H.
      • Stewart C.L.
      • Sullivan T.
      • Burghardt A.
      • Majumdar S.
      • Reue K.
      • et al.
      Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.
      ).
      Figure thumbnail gr11
      Fig. 11Accumulation of prelamin A at the nuclear envelope in Zmpste24−/− fibroblasts, as judged by confocal immunofluorescence microscopy. Normally, prelamin A is undetectable in cells; in the setting of Zmpste24 deficiency, abundant prelamin A is located at the nuclear envelope, and blebs are evident.
      Prelamin A and lamin C are products of the same gene (Lmna) and result from alternative splicing. The lamin C transcript terminates at the end of exon 10; the prelamin A transcript requires splicing from mid-exon 10 to exon 11, and then to exon 12 (
      • Lin F.
      • Worman H.J.
      Structural organization of the human gene encoding nuclear lamin A and nuclear lamin C.
      ). Lamins A and C are identical for 566 amino acids but then diverge at the C-terminal domains (
      • Mounkes L.C.
      • Burke B.
      • Stewart C.L.
      The A-type lamins. Nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.
      ,
      • Burke B.
      • Stewart C.L.
      Life at the edge: the nuclear envelope and human disease.
      ,
      • Fisher D.Z.
      • Chaudhary N.
      • Blobel G.
      cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins.
      ). Lamin A contains 98 lamin A-specific amino acids at its C terminus, including the CAAX motif; lamin C, which lacks a CAAX motif, contains 6 unique amino acids at its C terminus.
      Lamin B1 and lamin B2 contain nuclear localization motifs and are further targeted to the inner nuclear membrane by their C-terminal farnesylcysteine methyl ester (
      • Chelsky D.
      • Sobotka C.
      • O'Neill C.L.
      Lamin B methylation and assembly into the nuclear envelope.
      ,
      • Izumi M.
      • Vaughan O.A.
      • Hutchison C.J.
      • Gilbert D.M.
      Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells.
      ). Prelamin A is also targeted to the nucleus by a nuclear localization sequence; its farnesylcysteine methyl ester is important for targeting prelamin A to the inner nuclear membrane (
      • Beck L.A.
      • Hosick T.J.
      • Sinensky M.
      Isoprenylation is required for the processing of the lamin A precursor.
      ), where it almost certainly undergoes the second endoproteolytic processing step that releases mature lamin A (
      • Hennekes H.
      • Nigg E.A.
      The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties.
      ). [Zmpste24 is an ER protein (
      • Schmidt W.K.
      • Tam A.
      • Fujimura-Kamada K.
      • Michaelis S.
      Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage.
      ,
      • Kumagai H.
      • Kawamura Y.
      • Yanagisawa K.
      • Komano H.
      Identification of a human cDNA encoding a novel protein structurally related to the yeast membrane-associated metalloprotease, Ste24p.
      ), but we presume that continuities between the ER and the nuclear envelope mean that Zmpste24 may also be located along the inner nuclear membrane.] Noncleavable prelamin A mutants, which cannot undergo the second processing step, localize to the nuclear envelope (
      • Hennekes H.
      • Nigg E.A.
      The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties.
      ). Prelamin A mutations that abrogate farnesylation (i.e., mutants in which the C of the CAAX motif is changed to another amino acid such as serine) do not prevent targeting to the nucleus, but the mutant protein remains largely in the nucleoplasm and fails to reach the nuclear envelope (
      • Hennekes H.
      • Nigg E.A.
      The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties.
      ). Interestingly, one report has suggested that the ability of lamin C to reach the nuclear envelope is at least partially dependent on lamin A (
      • Vaughan O.A.
      • Alvarez-Reyes M.
      • Bridger J.M.
      • Broers J.L.V.
      • Ramaekers F.C.S.
      • Wehnert M.
      • Morris G.E.
      • Whitfield W.G.F.
      • Hutchison C.J.
      Both emerin and lamin C depend on lamin A for localization at the nuclear envelope.
      ).
      Why does nature go to the trouble of modifying the C terminus of prelamin A, given that this portion of the molecule is simply clipped off and degraded? The most obvious explanation is that each of the posttranslational modifications render the C terminus of CAAX proteins more hydrophobic, facilitating the initial targeting of the protein to the inner nuclear membrane (
      • Corrigan D.P.
      • Kuszczak D.
      • Rusinol A.E.
      • Thewke D.P.
      • Hrycyna C.A.
      • Michaelis S.
      • Sinensky M.S.
      Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24.
      ,
      • Dalton M.
      • Sinensky M.
      Expression systems for nuclear lamin proteins: farnesylation in assembly of nuclear lamina.
      ,
      • Kilic F.
      • Johnson D.A.
      • Sinensky M.
      Subcellular localization and partial purification of prelamin A endoprotease: an enzyme which catalyzes the conversion of farnesylated prelamin A to mature lamin A.
      ). Several groups have evaluated mutant prelamin A constructs lacking the last 18 amino acids of the protein (i.e., “mature lamin A” constructs). These constructs are defective in their ability to be targeted to the nuclear envelope (
      • Hennekes H.
      • Nigg E.A.
      The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties.
      ).

      LAMIN A AND LAMIN C ARE ASSOCIATED WITH MULTIPLE GENETIC DISEASES

      Lamins A and C have attracted great interest because LMNA mutations cause a host of different human genetic diseases (“laminopathies”), including Dunnigan-type familial partial lipodystrophy (FPLD) (
      • Hegele R.A.
      Familial partial lipodystrophy: a monogenic form of the insulin resistance syndrome.
      ,
      • Hegele R.A.
      Molecular basis of partial lipodystrophy and prospects for therapy.
      ,
      • Garg A.
      Lipodystrophies.
      ), Emery-Dreifuss muscular dystrophy (
      • Bonne G.
      • Di Barletta M.R.
      • Varnous S.
      • Bécane H-M.
      • Hammouda E-H.
      • Merlini L.
      • Muntoni F.
      • Greenberg C.R.
      • Gary F.
      • Urtizberea J-A.
      • et al.
      Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy.
      ), limb-girdle muscular dystrophy (
      • Wilson K.L.
      The nuclear envelope, muscular dystrophy and gene expression.
      ), familial cardiomyopathy with conduction system disease (
      • Vytopil M.
      • Benedetti S.
      • Ricci E.
      • Galluzzi G.
      • Russo A.D.
      • Merlini L.
      • Boriani G.
      • Gallina M.
      • Morandi L.
      • Politano L.
      • et al.
      Mutation analysis of the lamin A/C gene (LMNA) among patients with different cardiomuscular phenotypes.
      ), one form of Charcot-Marie-Tooth peripheral neuropathy (
      • De Sandre-Giovannoli A.
      • Chaouch M.
      • Kozlov S.
      • Vallat J-M.
      • Tazir M.
      • Kassouri N.
      • Szepetowski P.
      • Hammadouche T.
      • Vandenberghe A.
      • Stewart C.L.
      • et al.
      Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse.
      ), and mandibuloacral dysplasia (MAD) (
      • Novelli G.
      • Muchir A.
      • Sangiuolo F.
      • Helbling-Leclerc A.
      • D'Apice M.R.
      • Massart C.
      • Capon F.
      • Sbraccia P.
      • Federici M.
      • Lauro R.
      • et al.
      Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C.
      ). In addition, LMNA mutations cause the classic human progeroid syndrome Hutchinson-Gilford progeria syndrome (HGPS) (
      • Eriksson M.
      • Brown W.T.
      • Gordon L.B.
      • Glynn M.W.
      • Singer J.
      • Scott L.
      • Erdos M.R.
      • Robbins C.M.
      • Moses T.Y.
      • Berglund P.
      • et al.
      Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome.
      ) and also atypical Werner's syndrome, a less severe progeroid syndrome that develops somewhat later in life (
      • Chen L.
      • Lee L.
      • Kudlow B.A.
      • Dos Santos H.G.
      • Sletvold O.
      • Shafeghati Y.
      • Botha E.G.
      • Garg A.
      • Hanson N.B.
      • Martin G.M.
      • et al.
      LMNA mutations in atypical Werner's syndrome.
      ,
      • Hegele R.A.
      Drawing the line in progeria syndromes.
      ). Most but not all of the laminopathies are dominantly inherited; most are also caused by missense mutations, although HGPS is caused by a splicing mutation (
      • Eriksson M.
      • Brown W.T.
      • Gordon L.B.
      • Glynn M.W.
      • Singer J.
      • Scott L.
      • Erdos M.R.
      • Robbins C.M.
      • Moses T.Y.
      • Berglund P.
      • et al.
      Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome.
      ) and a nonsense mutation in codon 6 of lamin A/C causes Emery-Dreifuss muscular dystrophy (
      • Bonne G.
      • Di Barletta M.R.
      • Varnous S.
      • Bécane H-M.
      • Hammouda E-H.
      • Merlini L.
      • Muntoni F.
      • Greenberg C.R.
      • Gary F.
      • Urtizberea J-A.
      • et al.
      Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy.
      ).
      FPLD is characterized by loss of adipose tissue in the extremities after puberty, increased adipose tissue in the neck and face, increased fasting and postprandial insulin levels, acanthosis nigricans, hypertriglyceridemia, reduced insulin-mediated glucose disposal, frank diabetes mellitus after age 20, and a heightened susceptibility to coronary artery disease (
      • Hegele R.A.
      Familial partial lipodystrophy: a monogenic form of the insulin resistance syndrome.
      ,
      • Hegele R.A.
      Molecular basis of partial lipodystrophy and prospects for therapy.
      ). Most of the mutations causing FPLD are located in exon 8, which encodes a portion of the C-terminal globular domain shared by lamins A and C (
      • Dhe-Paganon S.
      • Werner E.D.
      • Chi Y-I.
      • Shoelson S.E.
      Structure of the globular tail of nuclear lamin.
      ). Crystallographic studies have revealed that most FPLD mutations are confined to a small region on the surface of the C-terminal globular domain of lamin A/C, whereas mutations causing muscular dystrophy are located throughout the lamin A/C protein core and its surface (
      • Dhe-Paganon S.
      • Werner E.D.
      • Chi Y-I.
      • Shoelson S.E.
      Structure of the globular tail of nuclear lamin.
      ). Very similar findings were observed by Krimm et al. (
      • Krimm I.
      • Ostlund C.
      • Gilquin B.
      • Couprie J.
      • Hossenlopp P.
      • Mornon J.P.
      • Bonne G.
      • Courvalin J.C.
      • Worman H.J.
      • Zinn-Justin S.
      The Ig-like structure of the C-terminal domain of lamin A/C, mutated in muscular dystrophies, cardiomyopathy, and partial lipodystrophy.
      ), who investigated lamin A/C structure by nuclear magnetic resonance. FPLD is not the only laminopathy characterized by loss of adipose tissue; this phenotype is also a prominent feature of MAD and HGPS.
      Several laminopathies exhibit a recessive inheritance pattern. Homozygosity for a LMNA R298C mutation causes some cases of Charcot-Marie-Tooth neuropathy type IIA (
      • De Sandre-Giovannoli A.
      • Chaouch M.
      • Kozlov S.
      • Vallat J-M.
      • Tazir M.
      • Kassouri N.
      • Szepetowski P.
      • Hammadouche T.
      • Vandenberghe A.
      • Stewart C.L.
      • et al.
      Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse.
      ), a motor and sensory neuropathy characterized by muscle weakness, secondary foot deformities, a slight reduction in nerve conduction velocities, with the loss of large myelinated fibers and axonal degeneration. MAD is caused by homozygosity for R527H (
      • Novelli G.
      • Muchir A.
      • Sangiuolo F.
      • Helbling-Leclerc A.
      • D'Apice M.R.
      • Massart C.
      • Capon F.
      • Sbraccia P.
      • Federici M.
      • Lauro R.
      • et al.
      Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C.
      ) or A529V substitutions (
      • Garg A.
      • Cogulu O.
      • Ozkinay F.
      • Onay H.
      • Agarwal A.K.
      A novel homozygous Ala529Val LMNA mutation in Turkish patients with mandibuloacral dysplasia.
      ). MAD is a progeroid syndrome characterized by postnatal growth retardation, lipodystrophy, insulin resistance, stiff joints, alopecia, abnormalities in the cranial sutures, osteolysis of the digits, shortening of the clavicle, micrognathia, and reduced ossification of alveolar bone.
      Some but not all lamin A/C missense mutations cause abnormalities in the shape and structure of the nuclear envelope, resulting in misshapen nuclei (
      • Muchir A.
      • Medioni J.
      • Laluc M.
      • Massart C.
      • Arimura T.
      • van der Kooi A.J.
      • Desguerre I.
      • Mayer M.
      • Ferrer X.
      • Briault S.
      • et al.
      Nuclear envelope alterations in fibroblasts from patients with muscular dystrophy, cardiomyopathy, and partial lipodystrophy carrying lamin A/C gene mutations.
      ). However, the mechanisms by which the lamin A/C mutations cause such a diverse collection of human diseases remain fairly mysterious. Presumably, the explanation relates to abnormalities in heterochromatin organization, the binding of nuclear factors and transcription factors (
      • Wilson K.L.
      • Zastrow M.S.
      • Lee K.K.
      Lamins and disease: insights into nuclear infrastructure.
      ), and secondary effects on gene expression (
      • Mounkes L.C.
      • Burke B.
      • Stewart C.L.
      The A-type lamins. Nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.
      ,
      • Wilson K.L.
      The nuclear envelope, muscular dystrophy and gene expression.
      ,
      • Hutchison C.J.
      • Alvarez-Reyes M.
      • Vaughan O.A.
      Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes?.
      ). It has been commonly suggested that muscle and heart disease with lamin A/C mutations could be attributable to the motion-related fragility of the nuclear envelope (
      • Mounkes L.C.
      • Burke B.
      • Stewart C.L.
      The A-type lamins. Nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.
      ,
      • Wilson K.L.
      The nuclear envelope, muscular dystrophy and gene expression.
      ,
      • Hutchison C.J.
      • Alvarez-Reyes M.
      • Vaughan O.A.
      Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes?.
      ). Recently, Lammerding and coworkers (
      • Lammerding J.
      • Schulze P.C.
      • Takahashi T.
      • Kozlov S.
      • Sullivan T.
      • Kamm R.D.
      • Stewart C.L.
      • Lee R.T.
      Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.
      ) as well as Broers et al. (
      • Broers J.L.
      • Peeters E.A.
      • Kuijpers H.J.
      • Endert J.
      • Bouten C.V.
      • Oomens C.W.
      • Baaijens F.P.
      • Ramaekers F.C.
      Decreased mechanical stiffness in LMNA−/− cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies.
      ) reported data in support of this concept. Lammerding et al. (
      • Lammerding J.
      • Schulze P.C.
      • Takahashi T.
      • Kozlov S.
      • Sullivan T.
      • Kamm R.D.
      • Stewart C.L.
      • Lee R.T.
      Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.
      ) used microscopic measurements and custom-built cell strain devices to demonstrate that lamin A/C knockout fibroblasts (Lmna−/−) have impaired nuclear mechanics and mechanotransduction properties (
      • Lammerding J.
      • Schulze P.C.
      • Takahashi T.
      • Kozlov S.
      • Sullivan T.
      • Kamm R.D.
      • Stewart C.L.
      • Lee R.T.
      Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.
      ). The nuclei from Lmna−/− fibroblasts exhibited exaggerated nuclear deformations, compared with Lmna+/+ control fibroblasts, when subjected to biaxial strain. Also, the nuclear envelope in Lmna−/− fibroblasts was unequivocally more fragile than in Lmna+/+ fibroblasts, as judged by experiments in which a 70 kDa dextran was microinjected into cell nuclei. Perhaps most intriguingly, the Lmna−/− fibroblasts exhibited significantly more cell death than Lmna+/+ fibroblasts when subjected to prolonged cyclic biaxial strain (
      • Lammerding J.
      • Schulze P.C.
      • Takahashi T.
      • Kozlov S.
      • Sullivan T.
      • Kamm R.D.
      • Stewart C.L.
      • Lee R.T.
      Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.
      ). Dual labeling with propidium iodide and an FITC-conjugated annexin V antibody revealed that the decrease in cell viability was caused by increased numbers of both necrotic and apoptotic cells. Lammerding et al. (
      • Lammerding J.
      • Schulze P.C.
      • Takahashi T.
      • Kozlov S.
      • Sullivan T.
      • Kamm R.D.
      • Stewart C.L.
      • Lee R.T.
      Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.
      ) also found diminished expression of the mechanosensitive genes egr-1 and the antiapoptotic gene iex-1 in Lmna−/− fibroblasts in response to mechanical stimulation.
      To date, no mutations in lamin B1 or lamin B2 have been linked to human disease, but it would not be particularly surprising if such associations were eventually identified. Vergnes and coworkers (
      • Vergnes L.
      • Peterfy M.
      • Bergo M.O.
      • Young S.G.
      • Reue K.
      Lamin B1 is required for mouse development and nuclear integrity.
      ) characterized mice lacking a functional lamin B1 (Lmnb1−/−). Homozygous Lmnb1−/− mice survived embryonic development but died at birth with defects in lung and bone. Of note, Lmnb1−/− fibroblasts displayed grossly misshapen nuclei, impaired differentiation, increased polyploidy, and premature senescence (
      • Vergnes L.
      • Peterfy M.
      • Bergo M.O.
      • Young S.G.
      • Reue K.
      Lamin B1 is required for mouse development and nuclear integrity.
      ).

      LAMIN A/C KNOCKOUT MICE

      Although Lmnb1 is expressed in all cells, Lmna is expressed late in development and then only in differentiated cells (
      • Mounkes L.C.
      • Burke B.
      • Stewart C.L.
      The A-type lamins. Nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.
      ,
      • Sullivan T.
      • Escalante-Alcalde D.
      • Bhatt H.
      • Anver M.
      • Bhat N.
      • Nagashima K.
      • Stewart C.L.
      • Burke B.
      Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.
      ). This interesting expression pattern prompted Sullivan and coworkers (
      • Sullivan T.
      • Escalante-Alcalde D.
      • Bhatt H.
      • Anver M.
      • Bhat N.
      • Nagashima K.
      • Stewart C.L.
      • Burke B.
      Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.
      ) in the laboratory of Dr. Colin Stewart to generate Lmna−/− mice. Lmna−/− mice survive for ∼5–6 weeks but then succumb with prominent signs of muscular dystrophy (
      • Sullivan T.
      • Escalante-Alcalde D.
      • Bhatt H.
      • Anver M.
      • Bhat N.
      • Nagashima K.
      • Stewart C.L.
      • Burke B.
      Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.
      ). Microscopic analysis of sections of Lmna−/− muscle revealed small dystrophic myocytes, with “hyalin or flocculent cytoplasm,” and some myocyte apoptosis/necrosis (
      • De Sandre-Giovannoli A.
      • Chaouch M.
      • Kozlov S.
      • Vallat J-M.
      • Tazir M.
      • Kassouri N.
      • Szepetowski P.
      • Hammadouche T.
      • Vandenberghe A.
      • Stewart C.L.
      • et al.
      Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse.
      ). Also, the peripheral nerves of lamin A/C knockout mice are abnormal, with a reduction in axon density, an increase in axon diameter, and an increase in the number of nonmyelinated axons (
      • De Sandre-Giovannoli A.
      • Chaouch M.
      • Kozlov S.
      • Vallat J-M.
      • Tazir M.
      • Kassouri N.
      • Szepetowski P.
      • Hammadouche T.
      • Vandenberghe A.
      • Stewart C.L.
      • et al.
      Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse.
      ). Lmna−/− mice have reduced subcutaneous fat, but this could be attributable to stress or reduced nutrition rather than to a bona fide lipodystrophy, as they had no apparent abnormalities in insulin, glucose, or triglyceride metabolism (
      • Sullivan T.
      • Escalante-Alcalde D.
      • Bhatt H.
      • Anver M.
      • Bhat N.
      • Nagashima K.
      • Stewart C.L.
      • Burke B.
      Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.
      ,
      • Cutler D.A.
      • Sullivan T.
      • Marcus-Samuels B.
      • Stewart C.L.
      • Reitman M.L.
      Characterization of adiposity and metabolism in Lmna-deficient mice.
      ). The absence of lamin A/C caused grossly misshapen nuclei and structurally abnormal nuclear envelopes, both in liver cells and in cultured fibroblasts (
      • De Sandre-Giovannoli A.
      • Chaouch M.
      • Kozlov S.
      • Vallat J-M.
      • Tazir M.
      • Kassouri N.
      • Szepetowski P.
      • Hammadouche T.
      • Vandenberghe A.
      • Stewart C.L.
      • et al.
      Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse.
      ).
      An intriguing finding was that emerin, a nuclear envelope protein that binds to lamins A/C, was mislocalized to the ER in Lmna−/− fibroblasts, indicating that lamin A or C (or both) is required for its proper targeting to the nuclear envelope (
      • Sullivan T.
      • Escalante-Alcalde D.
      • Bhatt H.
      • Anver M.
      • Bhat N.
      • Nagashima K.
      • Stewart C.L.
      • Burke B.
      Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.
      ). Emerin is important for muscle physiology; the emerin gene is mutated in X-linked Emery-Dreifuss muscular dystrophy (
      • Burke B.
      • Stewart C.L.
      Life at the edge: the nuclear envelope and human disease.
      ). One group has reported that lamin A, but not lamin C, plays a key role in the targeting of emerin to the nuclear envelope (
      • Raharjo W.H.
      • Enarson P.
      • Sullivan T.
      • Stewart C.L.
      • Burke B.
      Nuclear envelope defects associated with LMNA mutations cause dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy.
      ). However, a group in France transfected both lamin A and lamin C cDNA constructs into lamin A/C-deficient fibroblasts, and they concluded that both lamin A and lamin C were capable of promoting the targeting of emerin to the nuclear envelope (
      • Muchir A.
      • van Engelen B.G.
      • Lammens M.
      • Mislow J.M.
      • McNally E.
      • Schwartz K.
      • Bonne G.
      Nuclear envelope alterations in fibroblasts from LGMD1B patients carrying nonsense Y259X heterozygous or homozygous mutation in lamin A/C gene.
      ). They also showed that another integral membrane protein, nesprin-1α, is also mislocalized to the ER in the absence of lamin A/C (
      • Muchir A.
      • van Engelen B.G.
      • Lammens M.
      • Mislow J.M.
      • McNally E.
      • Schwartz K.
      • Bonne G.
      Nuclear envelope alterations in fibroblasts from LGMD1B patients carrying nonsense Y259X heterozygous or homozygous mutation in lamin A/C gene.
      ).

      PROCESSING OF PRELAMIN A

      Protein sequencing of the C-terminal region of lamin A indicated that prelamin A must undergo endoproteolytic processing, with the release of the farnesylcysteine methyl ester (
      • Weber K.
      • Plessmann U.
      • Traub P.
      Maturation of nuclear lamin A involves a specific carboxy-terminal trimming, which removes the polyisoprenylation site from the precursor: implications for the structure of the nuclear lamina.
      ). Sinensky and coworkers (
      • Beck L.A.
      • Hosick T.J.
      • Sinensky M.
      Isoprenylation is required for the processing of the lamin A precursor.
      ) showed that the processing of prelamin A to lamin A is completely dependent on protein prenylation. The same group has also actively investigated the enzymatic activity responsible for clipping off the last 15 amino acids from farnesyl-prelamin A (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ). They developed an assay for the endoprotease with a peptide substrate corresponding to the last 18 amino acids of the protein (ending with the methylated farnesylcysteine) (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ). They found that the prelamin A endoprotease was present in crude nuclear extracts and that the enzyme was entirely specific for a prelamin A peptide containing an S-farnesylated cysteine methyl ester (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ). The endoprotease was inhibited by N-acetyl farnesylcysteine, and the removal of the methyl ester from the substrate reduced enzymatic activity (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ). Inhibitor studies suggested that the prelamin A endoprotease activity may have been a serine protease (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ,
      • Kilic F.
      • Johnson D.A.
      • Sinensky M.
      Subcellular localization and partial purification of prelamin A endoprotease: an enzyme which catalyzes the conversion of farnesylated prelamin A to mature lamin A.
      ). Dr. Sinensky's laboratory has developed several lines of evidence indicating that the biochemical activity is located in the nucleus (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ,
      • Kilic F.
      • Johnson D.A.
      • Sinensky M.
      Subcellular localization and partial purification of prelamin A endoprotease: an enzyme which catalyzes the conversion of farnesylated prelamin A to mature lamin A.
      ), and this concept has been supported by others (
      • Hennekes H.
      • Nigg E.A.
      The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties.
      ).
      In yeast, a-factor is cleaved twice by Ste24p (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ). Simply because prelamin A in mammals is also cleaved twice, we hypothesized that prelamin A might be a substrate for Zmpste24 (
      • Young S.G.
      • Ambroziak P.
      • Kim E.
      • Clarke S.
      Postisoprenylation protein processing: CXXX (CaaX) endoproteases and isoprenylcysteine carboxyl methyltransferase.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ). At the time, this hypothesis seemed extremely far-fetched, given that prelamin A had no obvious structural similarities to yeast a-factor. In any case, to approach this issue experimentally, we generated Zmpste24−/− mice (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ). Along with another group that also generated Zmpste24−/− mice (
      • Pendás A.M.
      • Zhou Z.
      • Cadiñanos J.
      • Freije J.M.P.
      • Wang J.
      • Hultenby K.
      • Astudillo A.
      • Wernerson A.
      • Rodríguez F.
      • Tryggvason K.
      • et al.
      Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice.
      ), we showed that Zmpste24−/− fibroblasts manifested a striking defect in prelamin A processing (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ), with an accumulation of prelamin A and a complete absence of mature lamin A (Fig. 10). With an antibody specific for the C terminus of prelamin A (the segment of prelamin A that is normally cleaved off), we observed a striking accumulation of a 74 kDa prelamin A in Zmpste24−/− cells. In extracts from wild-type cells, prelamin A is virtually undetectable, for the simple reason that processing of prelamin A to mature lamin A is an extremely efficient process, rapidly converting the prelamin A to mature lamin A. Western blots of Zmpste24−/− extracts with an antibody against the N terminus of lamin A/C revealed lamin C and prelamin A (74 kDa) but absolutely no mature lamin A.
      An FTI completely blocked the processing of prelamin A to mature lamin A (
      • Bergo M.O.
      • Gavino B.
      • Ross J.
      • Schmidt W.K.
      • Hong C.
      • Kendall L.V.
      • Mohr A.
      • Meta M.
      • Genant H.
      • Jiang Y.
      • et al.
      Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.
      ), a result that was consistent with earlier studies from the laboratory of Sinensky (
      • Beck L.A.
      • Hosick T.J.
      • Sinensky M.
      Isoprenylation is required for the processing of the lamin A precursor.
      ). We also asked whether prelamin A might accumulate in fibroblasts lacking Icmt (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.J.
      • Gomes A.Q.
      • Seabra M.C.
      • Young S.G.
      Isoprenylcysteine carboxyl methyltransferase deficiency in mice.
      ) or Rce1 (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ). In our initial experiments with Icmt−/− fibroblasts, prelamin A processing appeared to be fully blocked. That result was consistent with the earlier biochemical studies from Sinensky and colleagues (
      • Kilic F.
      • Dalton M.B.
      • Burrell S.K.
      • Mayer J.P.
      • Patterson S.D.
      • Sinensky M.
      In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
      ) indicating that the C-terminal methyl ester was essential for the endoproteolytic processing reaction. However, our recent experiments with several immortalized Icmt−/− cell lines have shown that the defect in prelamin A processing is only partial (
      • Young S.G.
      • Clarke S.
      • Bergo M.
      • Philips M.
      • Fong L.G.
      Genetic approaches to understanding the physiologic importance of the carboxyl methylation of isoprenylated proteins.
      ). We invariably observe an increase in the amount of prelamin A in Icmt−/− cell lines, as judged by Western blots with an antibody against the C terminus of prelamin A (Fig. 12). Thus, the prelamin A processing in Icmt−/− fibroblasts is clearly abnormal. However, when Western blotting is performed with an antibody against the N-terminal portion of lamin A/C, we always observe some mature lamin A. Thus, Icmt deficiency causes a partial but not a complete blockade in the processing of prelamin A to mature lamin A.
      Figure thumbnail gr12
      Fig. 12Increased amounts of prelamin A in fibroblasts lacking Icmt. A: Western blot of wild-type, Zmpste24−/−, and Icmt−/− fibroblasts with a prelamin A-specific antibody and an antibody against β-actin. B: Western blot of Zmpste24−/− and Icmt−/− fibroblasts with a lamin A/C-specific antibody. Reproduced, with permission, from The Enzymes (
      • Young S.G.
      • Clarke S.
      • Bergo M.
      • Philips M.
      • Fong L.G.
      Genetic approaches to understanding the physiologic importance of the carboxyl methylation of isoprenylated proteins.
      ).
      Prelamin A processing in Rce1−/− fibroblasts was normal (
      • Bergo M.O.
      • Gavino B.
      • Ross J.
      • Schmidt W.K.
      • Hong C.
      • Kendall L.V.
      • Mohr A.
      • Meta M.
      • Genant H.
      • Jiang Y.
      • et al.
      Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.
      ). One interpretation of this finding is that Rce1 plays absolutely no role in prelamin A processing (
      • Agarwal A.K.
      • Fryns J-P.
      • Auchus R.J.
      • Garg A.
      Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia.
      ). However, we believe that this interpretation is unlikely. In yeast, the cleavage of the AAX from a-factor is a redundant function of Rce1p and Ste24p (
      • Chen P.
      • Sapperstein S.K.
      • Choi J.D.
      • Michaelis S.
      Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.
      ,
      • Boyartchuk V.L.
      • Rine J.
      Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.
      ), and we suspect that both Rce1 and Zmpste24 play redundant roles in cleaving the AAX from prelamin A (
      • Bergo M.O.
      • Gavino B.
      • Ross J.
      • Schmidt W.K.
      • Hong C.
      • Kendall L.V.
      • Mohr A.
      • Meta M.
      • Genant H.
      • Jiang Y.
      • et al.
      Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.
      ). No proof for this view yet exists, but several “soft” observations tend to support it. First, Rce1 processes a very broad range of mammalian CAAX proteins (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.
      ,
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      Cloning and characterization of a mammalian prenyl protein-specific protease.
      ), and there is no reason to believe that it would not be capable of cleaving prelamin A. Rce1 is exclusively responsible for cleaving the AAX from another lamin, lamin B1 (
      • Maske C.P.
      • Hollinshead M.S.
      • Higbee N.C.
      • Bergo M.O.
      • Young S.G.
      • Vaux D.J.
      A carboxyl-terminal interaction of lamin B1 is dependent on the CAAX endoprotease Rce1 and carboxymethylation.
      ), in which the CAAX motif (CAIM) is similar to that in prelamin A (CSIM). Second, the prelamin A CAAX motif is actually a better substrate for yeast Rce1p than yeast Ste24p (Fig. 5), and the specificities of the mouse Zmpste24 enzymes resemble those of yeast Ste24p (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ).
      The precise biochemical role of Zmpste24 in prelamin A processing is beginning to come into focus. Corrigan and coworkers (
      • Corrigan D.P.
      • Kuszczak D.
      • Rusinol A.E.
      • Thewke D.P.
      • Hrycyna C.A.
      • Michaelis S.
      • Sinensky M.S.
      Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24.
      ) have presented evidence that recombinant ZMPSTE24, produced in insect cells, is capable of carrying out both the release of the AAX from a short prelamin A peptide and the release of the 15 additional amino acids. The ability of Zmpste24 to clip the AAX from prelamin A parallels the ability of Ste24p to cleave the AAX from a-factor (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      Modulation of Ras and a-factor function by carboxyl-terminal proteolysis.
      ). However, we suspect that Zmpste24's ability to act as a CAAX endoprotease beyond prelamin A is very restricted. We tested whether recombinant Zmpste24 would be capable of processing some or all of the CAAX protein substrates that accumulate in Rce1−/− fibroblasts (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ). Whole cell extracts from Rce1+/+ and Rce1−/− fibroblasts were incubated with recombinant Icmt, S-adenosyl-l-[methyl-14C]methionine, and either recombinant mouse Rce1 or Zmpste24. We noted significantly increased carboxyl methylation when recombinant Rce1 was added to extracts from Rce1−/− fibroblasts, reflecting the processing of accumulated Rce1 substrates (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ). No such increase was observed when recombinant Zmpste24 was added to extracts from Rce1−/− fibroblasts (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S.
      • Young S.G.
      Biochemical studies of Zmpste24-deficient mice.
      ), indicating that Zmpste24 has little if any ability to process the substrates that accumulate in Rce1−/− fibroblasts.
      Zmpste24−/− cells contain a striking accumulation of prelamin A. One would expect that prelamin A would be farnesylated in Zmpste24−/− cells, because farnesylation precedes the endoproteolytic cleavage step mediated by Zmpste24. Indeed, several lines of evidence suggest that it actually is farnesylated. First, the electrophoretic migration of prelamin A in human and mouse Zmpste24−/− fibroblasts is retarded when cells are grown in the presence of an FTI (
      • Toth J.I.
      • Yang S.H.
      • Qiao X.
      • Beigneux A.P.
      • Gelb M.H.
      • Moulson C.L.
      • Miner J.H.
      • Young S.G.
      • Fong L.G.
      Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.
      ). Also, the prelamin A in Zmpste24−/− fibroblasts is located at the nuclear envelope (Fig. 11) (
      • Fong L.G.
      • Ng J.K.
      • Meta M.
      • Cote N.
      • Yang S.H.
      • Stewart C.L.
      • Sullivan T.
      • Burghardt A.
      • Majumdar S.
      • Reue K.
      • et al.
      Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.
      ,
      • Pendás A.M.
      • Zhou Z.
      • Cadiñanos J.
      • Freije J.M.P.
      • Wang J.
      • Hultenby K.
      • Astudillo A.
      • Wernerson A.
      • Rodríguez F.
      • Tryggvason K.
      • et al.
      Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice.
      ,
      • Yang S.H.
      • Bergo M.O.
      • Toth J.I.
      • Qiao X.
      • Hu Y.
      • Sandoval S.
      • Meta M.
      • Bendale P.
      • Gelb M.H.
      • Young S.G.
      • et al.
      Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts containing a targeted Hutchinson-Gilford progeria syndrome mutation.
      ), whereas nonfarnesylated prelamin A is misdirected to the nucleoplasm (
      • Yang S.H.
      • Bergo M.O.
      • Toth J.I.
      • Qiao X.
      • Hu Y.
      • Sandoval S.
      • Meta M.
      • Bendale P.
      • Gelb M.H.
      • Young S.G.
      • et al.
      Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts containing a targeted Hutchinson-Gilford progeria syndrome mutation.
      ).
      As explained in more detail below, FTIs have recently been used to improve the nuclear shape in progeroid disorders characterized by an accumulation of farnesylated prelamin A at the nuclear envelope (
      • Toth J.I.
      • Yang S.H.
      • Qiao X.
      • Beigneux A.P.
      • Gelb M.H.
      • Moulson C.L.
      • Miner J.H.
      • Young S.G.
      • Fong L.G.
      Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.
      ,
      • Yang S.H.
      • Bergo M.O.
      • Toth J.I.
      • Qiao X.
      • Hu Y.
      • Sandoval S.
      • Meta M.
      • Bendale P.
      • Gelb M.H.
      • Young S.G.
      • et al.
      Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts containing a targeted Hutchinson-Gilford progeria syndrome mutation.
      ). The rationale is that inhibition of farnesylation would mislocalize farnesyl-prelamin A away from the nuclear envelope, thereby attacking the presumptive cause of the misshapen nuclei. However, we suggest that this may not be the only pharmacological strategy. As noted previously, methylation appears to be critical for the membrane attachment of farnesylated proteins (
      • Michaelson D.
      • Ali W.
      • Chiu V.K.
      • Bergo M.
      • Silletti J.
      • Wright L.
      • Young S.G.
      • Philips M.
      Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases.
      ,
      • Silvius J.R.
      • l'Heureux F.
      Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers.
      ), and we would not be surprised if Icmt inhibitors, currently under development (
      • Anderson J.L.
      • Henriksen B.S.
      • Gibbs R.A.
      • Hrycyna C.A.
      The isoprenoid substrate specificity of isoprenylcysteine carboxylmethyltransferase: development of novel inhibitors.
      ,
      • Winter-Vann A.M.
      • Casey P.J.
      Post-prenylation-processing enzymes as new targets in oncogenesis.
      ,
      • Winter-Vann A.M.
      • Baron R.A.
      • Wong W.
      • Cruz J. dela
      • York J.D.
      • Gooden D.M.
      • Bergo M.O.
      • Young S.G.
      • Toone E.J.
      • Casey P.J.
      A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells.
      ), might work independently or synergistically to mislocalize farnesyl-prelamin A within the nucleus.

      ZMPSTE24 KNOCKOUT MICE

      Mice lacking Zmpste24, initially developed by Leung and coworkers (
      • Leung G.K.
      • Schmidt W.K.
      • Bergo M.O.
      • Gavino B.
      • Wong D.H.
      • Tam A.
      • Ashby M.N.
      • Michaelis S