Originally published In Press as doi:10.1194/jlr.R500011-JLR200 on October 5, 2005
Journal of Lipid Research, Vol. 46, 2531-2558, December 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology
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
Stephen G. Young1,*,
Loren G. Fong* and
Susan Michaelis1,
* Division of Cardiology, Department of Internal Medicine, University of California, Los Angeles, CA 90095
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205
Published, JLR Papers in Press, October 5, 2005. DOI 10.1194/jlr.R500011-JLR200
1 To whom correspondence should be addressed. e-mail: sgyoung{at}mednet.ucla.edu (S.G.Y.); michaelis{at}jhmi.edu (S.M.)
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ABSTRACT
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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.
Supplementary key words protein prenylation • laminopathy • aging • Ste24 • a-factor • lamin A/C
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INTRODUCTION
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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 (1–4). 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 (5–8). 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 (1, 2, 9, 10). 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 (11–14). 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 (15, 16). 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 (17–24). 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.
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POSTTRANSLATIONAL MODIFICATIONS OF CAAX PROTEINS
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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 (25, 26). The CAAX sequence triggers three sequential enzymatic modifications (Fig. 1)
(1, 26). 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 (1, 26). In general, the cysteine is geranylgeranylated if the X is a leucine or phenylalanine; otherwise, it is farnesylated (1, 27). 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 (1). After endoproteolysis, the newly exposed isoprenylcysteine is methylated by an ER membrane prenylprotein-specific methyltransferase, isoprenylcysteine carboxyl methyltransferase (Icmt, designated Ste14p in yeast) (28, 29).
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Fig. 1. Posttranslational 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.
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The CAAX motif modifications render the C terminus of the protein more hydrophobic, facilitating the binding of proteins to membrane surfaces (17, 30). 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) (9, 17, 31, 32), enhance the metabolic stability of proteins (a-factor intermediates in yeast lacking FTase or Ste14p are degraded rapidly) (17, 32), and facilitate the targeting of proteins to specific sites within the cell (20, 33–36). For example, in yeast, Ras proteins that do not undergo endoproteolysis or carboxyl methylation are not properly localized to the plasma membrane (Fig. 2)
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PROCESSING OF MATING PHEROMONES IN S. CEREVISIAE
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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) (31, 37–39). The A-10 mating factor of T. mesenterica was found to contain an 
-carboxyl methyl ester of a C-terminal cysteine residue, which was also modified by an S-farnesyl group (37, 38). Similar findings were made on the A(Ia) mating factor of T. brasiliensis (40, 41). 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 MAT, which secrete the diffusible signaling molecules, a-factor and -factor, respectively (42). 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 -factor, the two pheromones are structurally and biochemically distinct. -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 (18, 43–45).
Studies from our laboratory (17, 18, 21, 46–49) and others (19, 20) 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.
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Fig. 4. Scheme 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 (17, 18, 47, 48, 57). 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 (49).
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IDENTIFICATION OF ENZYMES RESPONSIBLE FOR THE C-TERMINAL PROCESSING OF a-FACTOR
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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) (23, 44). The identification of RAM1 (named for Ras and a-factor maturation) in entirely different genetic screens suggested a commonality between Ras and a-factor (23, 50). 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 (24).
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, ste14
, that lacked the capacity to produce a functional a-factor (21, 22, 32). Because Ste14p is an integral membrane protein of the ER membrane, the purification of the enzyme presented major challenges. However, heterologous expression studies (21, 51), and ultimately biochemical experiments with a purified Ste14p enzyme (52), 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 (20). 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 (20) 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 (20). 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 (53). Mutating either of the conserved histidines in the HEXXH domain blocked the ability of Ste24p to complement the mating defect of STE24-deficient yeast (ste24) expressing the CAMQ form of a-factor (20).
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 ste24 yeast membranes was insensitive to the zinc chelator 1,10-o-phenanthroline, suggesting that the remaining endoprotease was not a zinc metalloprotease (20). To isolate the remaining endoprotease, ste24 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 (20). 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 (20). Yeast lacking RCE1 (rce1) manifest a modest decrease in a-factor production, and membranes from rce1 yeast had moderately decreased CAAX endoprotease activity in in vitro assays with farnesylated a-factor peptide substrates. The residual CAAX endoprotease activity in rce1 yeast was sensitive to o-phenanthroline, consistent with Ste24p being a zinc metalloprotease (20).
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)
(20, 54–56). 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 -mating type. The single mutants (ste24 and rce1) have modestly reduced a-factor production and therefore yield slightly smaller halos, whereas the double mutant (ste24rce1) 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).
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Fig. 5. The 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 (ste24 or rce1), or both (ste24rce1) 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 MAT 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 (20, 55); CAMQ from the mammalian phosphorylase kinase subunit, which is Ste24p-specific (20); CVIA, the a-factor CAAX motif, which can be cleaved by either Rce1p or Ste24p (19, 20, 47); and CSIM, the prelamin CAAX motif, which appears to be Rce1p-specific (M. Boyle and S. Michaelis, unpublished data).
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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) (19). 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 (55). 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.
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A SECOND ROLE FOR STE24 IN THE BIOGENESIS OF a-FACTOR
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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) (18–20, 47). 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 (17). 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 (18). The second cleavage reaction, which coverts the P2 intermediate to mature a-factor, involves the release of an additional 14 amino acids (17, 43).
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) (17). 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 (57). In axl1 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 (axl1ste23) are completely sterile (57).
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 (18). 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 (18), followed soon thereafter by the report demonstrating the role of Ste24p in the release of the AAX (20). 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 (19, 47). 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 (18, 19). 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.
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 ram1 or ram2 mutants (17, 24). 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 (32). 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 (49). 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 (49).
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THE STE24 ORTHOLOG IN MAMMALS, ZMPSTE24, COMPLEMENTS STE24 DEFICIENCY IN YEAST
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The genes encoding the mammalian postisoprenylation enzymes (ZMPSTE24, RCE1, and ICMT) were identified by homology with their yeast counterparts (STE24, RCE1, and STE14, respectively) (29, 47, 58). 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 (47). This point is illustrated in Fig. 7
, in which complementation of ste24rce1 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 ste24rce1 yeast (see Fig. 13A below) (54). 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 (59).
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Fig. 7. The human STE24 homolog, ZMPSTE24, complements the a-factor biosynthesis, halo, and mating defects when expressed in ste24rce1 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 (ste24rce1) 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) (47, 49). In the mating test, patches of auxotrophic MATa cells are replica-plated to a lawn of MAT 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 (47) and The Journal of Biological Chemistry (48) and is reproduced with permission.
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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 (18), the gene was later called AFC1 (for a-factor-converting enzyme) (20). 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) (47). Another group called it HsSte24 (60). 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 (61). 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.
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PHYSIOLOGIC IMPORTANCE OF RCE1 AND ICMT IN MAMMALS AND MICE
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Deciphering the physiologic importance of CAAX processing in mammals has been greatly aided by the development of Rce1 and Icmt knockout mice (35, 62). Nearly all homozygous knockout mice (Rce1–/–) died fairly late in gestation, beginning at embryonic day 14.5–15.5 (35). 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 (35).
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 (35). 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 (35).
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 (35, 58). In contrast, membranes from Rce1–/– fibroblasts did not process any of the Ras proteins. Thus, as in yeast (20), the endoproteolytic processing of the Ras proteins requires Rce1. The endoproteolytic processing of other CAAX proteins was also blocked (35, 58). Interestingly, Rce1 is required for the endoproteolytic processing of lamin B1. Maske et al. (63) 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 (54).
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 (35, 36). In contrast, the Ras proteins in Rce1–/– fibroblasts were strikingly mislocalized to the cytosol and internal membrane compartments (35, 36), 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 (64). 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 (64).
We also created mice lacking Icmt (33) 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 (65). 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 (62).
We were successful in culturing Icmt–/– fibroblasts from E11.5 mouse embryos (62). Icmt–/– fibroblasts lacked methyltransferase activity against both small-molecule Icmt substrates (e.g., N-acetyl-farnesylcysteine) and farnesylated proteins such as K-Ras (33). 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) (33). Icmt is the only enzyme for methylating prenylcysteines in mammalian cells, methylating both CAAX proteins and the CXC subset (62, 66) 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)
(36, 62). Recent studies have shown that methylation is important for CAAX protein targeting within cells when the protein is farnesylated (36). When the CAAX protein is geranylgeranylated, the contribution of methylation to intracellular targeting appears to be negligible or nonexistent (36, 67).
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Fig. 8. Mislocalization of K-Ras in spontaneously immortalized Icmt–/– fibroblasts that had been transfected with a GFP-K-Ras fusion construct (33, 34). The Ras proteins are similarly mislocalized in Rce1–/– fibroblasts (35, 36). Several of these images have been published previously in The Journal of Clinical Investigation (34) and The Enzymes (115) and are reproduced with permission.
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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 (34). The latter finding has prompted interest in Icmt inhibitors as possible anticancer agents (68–70).
Previous pharmacologic studies with inhibitors of protein farnesyltransferase had shown that farnesylation is critical for the proper intracellular targeting of CAAX proteins (71–74). Studies with Rce1- and Icmt-deficient fibroblasts (9, 33–36, 62) 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.
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NUCLEAR LAMINS: ABUNDANT MAMMALIAN CAAX PROTEINS
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Lamin A, lamin C, lamin B1, and lamin B2 are intermediate filament proteins and are among the most abundant CAAX proteins in mammalian cells (1, 75–77). 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 (12, 14). 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 (12, 78). 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 (79–81). As noted previously, however, prelamin A undergoes a second endoproteolytic processing step (82). 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 (81). In the absence of that enzyme, farnesyl-prelamin A accumulates within cells (Fig. 10)
, specifically at the nuclear envelope (Fig. 11)
(16, 83).
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Fig. 9. Biogenesis 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 (18–20, 47), 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 (63). Blocking farnesylation with a farnesyltransferase inhibitor (FTI) would mean that the C terminus of progerin would terminate with an -carboxylate anion rather than a farnesylcysteine methyl ester; this change would reduce the hydrophobicity of the C terminus of the protein (67) 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.
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Fig. 10. Western 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 (16).
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Fig. 11. Accumulation 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.
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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 (11). Lamins A and C are identical for 566 amino acids but then diverge at the C-terminal domains (12, 13, 84). 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 (85, 86). 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 (87), where it almost certainly undergoes the second endoproteolytic processing step that releases mature lamin A (88). [Zmpste24 is an ER protein (46, 60), 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 (88). 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 (88). Interestingly, one report has suggested that the ability of lamin C to reach the nuclear envelope is at least partially dependent on lamin A (89).
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 (81, 90, 91). 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 (88).
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LAMIN A AND LAMIN C ARE ASSOCIATED WITH MULTIPLE GENETIC DISEASES
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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) (92–94), Emery-Dreifuss muscular dystrophy (95), limb-girdle muscular dystrophy (78), familial cardiomyopathy with conduction system disease (96), one form of Charcot-Marie
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ABSTRACT
INTRODUCTION
