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Thematic Review |
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536-0509
Published, JLR Papers in Press, February 23, 2008.
1 To whom correspondence should be addressed. e-mail: bobd{at}uky.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSPHINGOLIPID METABOLISMCELLULAR PROCESSES REGULATED BY...SECRETORY PATHWAY, ENDOCYTOSIS,...SUMMARY AND FUTURE DIRECTIONSREFERENCES |
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Supplementary key words long-chain base • signal transduction • heat stress • actin • Target Of Rapamycin • phytosphingosine • very-long-chain fatty acids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSPHINGOLIPID METABOLISMCELLULAR PROCESSES REGULATED BY...SECRETORY PATHWAY, ENDOCYTOSIS,...SUMMARY AND FUTURE DIRECTIONSREFERENCES |
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The common baker's yeast Saccharomyces cerevisiae has served in many ways to foster our understanding of sphingolipid metabolism and functions, beginning with the early work of Herbert Carter and his students in the 1950s and early 1960s, who played seminal roles in characterizing sphingolipid long-chain bases (LCBs) (6). This was followed by elucidation of the types of complex sphingolipids found in yeast cells and their route of synthesis by Lester and colleagues (reviewed in Refs. 7, 8). More recently, yeast has been used to identify nearly all of the genes that encode sphingolipid metabolic enzymes, and many of these were critical in identifying human homologs (9, 10). This review describes and integrates advances in understanding the sphingolipid metabolism and functions of S. cerevisiae during the past 2 years and focuses on the role of LCBs in signal transduction pathways that regulate growth, responses to stress, particularly heat stress, exocytosis of plasma membrane proteins, endocytosis, and actin cytoskeleton dynamics. Previous reviews should be consulted for topics that are not presented here and for more detailed coverage of areas only briefly touched upon in this review (8–14).
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SPHINGOLIPID METABOLISM |
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TOPABSTRACTINTRODUCTIONSPHINGOLIPID METABOLISMCELLULAR PROCESSES REGULATED BY...SECRETORY PATHWAY, ENDOCYTOSIS,...SUMMARY AND FUTURE DIRECTIONSREFERENCES |
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As is the case with other lipids, sphingolipid synthesis begins in the endoplasmic reticulum (ER), where serine palmitoyltransferase (SPT) condenses serine with a fatty acyl-CoA to yield 3-ketodihydrosphingosine (ketosphinganine) and CO2 (Fig. 1
). In yeast, SPT is a heterodimer made from the Lcb1 and Lcb2 proteins (reviewed in Ref. 19). These proteins are ubiquitous and have been found in all organisms that make sphingolipids: in a unique case, they have been found fused into a single gene in a viral genome (20). Yeast SPT has a third, small hydrophobic subunit, Tsc3, that is necessary for optimal enzyme activity and for growth at temperatures above 
30°C, but its function remains unknown and it is not found in mammals (21). Recently, mammals were shown to make two types of Lcb2 subunits, SPTLC2 and SPTLC3, whose concentrations vary in a tissue-specific manner (22). Moreover, mammalian SPT appears to be a large molecular weight complex, possibly containing four SPTLC1 monomers associated with four total copies of SPTLC2 and SPTLC3, whose ratio in the complex varies in different cell types (23). It is not clear at this time whether the yeast SPT exists in such a large complex.
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Impressive progress in understanding how fatty acids are elongated from C14–C18 to C26 in the ER by the very-long-chain fatty acid (VLCFA) synthetase complex has been made recently. Four reactions constitute the elongation cycle, catalyzed by a multienzyme complex that initiates with the condensation of malonyl-CoA with an acyl-CoA to form a 3-ketoacyl-CoA (Fig. 1, step 1). The 3-ketoacyl-CoA is reduced to 3-hydroxy acyl-CoA in the second step, followed by the dehydration of 3-hydroxy acyl-CoA to an enoyl intermediate in the third step. In the fourth and final step, the enoyl is reduced to yield an acyl-CoA that is two carbons longer than the starting compound. The Elo1 protein was shown early on to catalyze the elongation of C14 fatty acids to C16 (34). Two related proteins, Fen1 and Sur4, were subsequently discovered and shown to play a role in elongation up to C26 (35). Elo1, Fen1, and Sur4 belong to a family of elongase proteins (Elops) (36) that specify chain length and that were thought to catalyze the first reaction in the cycle. The second reaction was shown to be catalyzed by Ybr159w (37, 38), and the final reaction was thought to be catalyzed by Tsc13 (39).
The identity of the protein that catalyzed the third reaction was unknown, and it was unclear how fatty acid chain length was determined. This confusion has been clarified with the reconstitution of the yeast VLCFA synthetase complex in liposomes (40). These studies verified that one of two Elops (36), either Fen1 or Sur4 (35), catalyzes the first reaction and that the third, dehydration reaction is catalyzed by the newly discovered Phs1 protein (41). A seminal contribution of the reconstituted elongation system was to clarify the function of the Fen1 and Sur4 Elops and to suggest that each enzyme complex contains a single Elop. A VLCFA synthetase complex with Fen1 was shown to make C22 and C24 fatty acids, whereas a complex with Sur4 could make longer C26 fatty acids. Mutational analysis of Fen1 and Sur4 revealed that a transmembrane helix containing a lysine residue and facing the lumen of the ER determines fatty acid chain length. The lysine is hypothesized to act as part of a caliper mechanism to specify chain length (40).
Ceramides must be transported from the ER to the Golgi so that the polar head groups can be added. In yeast, there appears to be a vesicle and a nonvesicle transport system that seems to require direct contact between the ER and Golgi membranes (42). Mammals also use vesicle and nonvesicle ceramide transport. Nonvesicle transport is mediated by the CERT protein, which extracts ceramides from the lumenal surface of the ER and deposits them on the outer leaflet of the Golgi membrane (43, 44). No CERT-like protein has been found in yeast, but this could be attributable simply to a lack of amino acid sequence conservation.
Upon reaching the Golgi membrane, ceramide incorporates into the outer leaflet and then either flips spontaneously or is flipped enzymatically (it is not clear which mechanism is important) to the inner leaflet so that it is accessible to the enzymes that attach polar head groups, which are in the lumen. The first head group to be added to the C1 OH of ceramides is inositol phosphate. This reaction is catalyzed by inositol phosphorylceramide synthase (IPC synthase) (45, 46) and yields the first complex sphingolipid, inositol phosphoceramide (IPC) (Fig. 1). The second complex sphingolipid, mannose inositol phosphoceramide (MIPC), is made by the transfer of mannose from GDP-mannose onto the inositol 2-OH moiety of IPC. The enzyme inositol phosphoceramide mannosyl transferase catalyzes this reaction and has two forms. One contains the Csg1 and Csg2 proteins, and the other contains the Csh1 and Csg2 proteins. The Csg1 and Csh1 subunits appear to be the catalytic subunits, whereas Csg2 performs a regulatory function (47). The Csg2 protein binds Ca2+, and transcription of the CSG2 gene is stimulated by high levels of Ca2+ in culture medium, as is the conversion of IPC to MIPC (48). These results may partly explain why csg2 mutants were isolated in a screen for mutations that give a calcium-sensitive growth phenotype (49). In the presence of high calcium, cells may have to convert IPC to MIPC at a faster rate to grow, either because IPC becomes toxic to an essential function in the Golgi apparatus or the plasma membrane or because MIPC or its product mannose-(inositol-P)2-ceramide [M(IP)2C] becomes rate-limiting for an essential process. This connection between calcium and complex sphingolipid synthesis is fascinating and needs to be examined more thoroughly. The calcium-sensitive phenotype of csg1 and csg2 mutants has been of great value in identifying genes involved in sphingolipid metabolism (24) and, as described below in this review, has provided new insights into how sphingolipid synthesis is regulated.
The terminal yeast complex sphingolipid made in the Golgi apparatus is M(IP)2C, made by the transfer of a second inositol phosphate from phosphatidylinositol to MIPC. Complex sphingolipids made in the Golgi apparatus move by vesicle transport primarily to the plasma membrane (50). Readers seeking more information about ceramide transport and sphingolipid metabolism in the Golgi apparatus should consult a previous review (14).
Although mammalian cells must turn over or break down complex sphingolipids to survive and prevent the accumulation of toxic molecules that cause debilitating human diseases termed sphingolipidoses (51), it has only fairly recently become apparent that S. cerevisiae cells even break down complex sphingolipids, let alone that the breakdown plays any observable physiological role. Complex sphingolipids in yeast constitute 30% of the phosphorylated membrane lipids and nearly 7% of the mass of the plasma membrane (52). Thus, it was experimentally challenging to biochemically detect the breakdown of a small fraction of complex sphingolipids and even more difficult to determine where the breakdown was occurring in cells. This technical challenge can be conquered by yeast genetics.
Because of amino acid sequence similarity to mammalian sphingomyelinases, the ISC1 gene was examined and found to be a phospholipase C-type enzyme that cleaved polar head groups from yeast sphingolipids (53, 54). Isc1 appears to be the only enzyme in yeast with such activity, and at this time it is the only known enzyme that breaks down complex sphingolipids in yeast. A short summary of the previously described roles of Isc1 is presented as an introduction to its recently discovered roles in the regulation of sphingolipid synthesis and stress protection.
Isc1 may have a role in tolerance to sodium and lithium ions (55), but the mechanism is unclear. Isc1 activity is required for growth on a nonfermentable carbon source such as during the diauxic shift, implying a role in respiration/mitochondria (56). A mitochondrial connection is supported by the observation that Isc1 moves from the ER to the mitochondria as cells progress from fermentative to respiratory growth during the diauxic shift (57). During this time, the specific activity of the enzyme increases by 3- to 5-fold and phytoceramide increases by 4-fold, but only if Isc1 is active. Lipids enriched in mitochondria, phosphatidylglycerol, and cardiolipin activate and appear to be physiologically important regulators of enzyme activity (56). Isc1 in cells grown to saturation (postdiauxic shift) was recently shown to reside in the outer leaflet of the mitochondrial membrane (58). Lipid analysis of purified mitochondria indicates a high content of 
-hydroxy-fatty acyl-containing phytoceramides, suggesting that it is derived from complex sphingolipids by the action of Isc1. Cells with an intact ISC1 gene are less likely than isc1
cells to lose mitochondrial function and become incapable of using nonfermentable carbon sources. Likewise, cells with wild-type Isc1 activity are more resistant to oxidative stress as measured by hydrogen peroxide resistance, and they resist ethidium bromide-induced mitochondrial damage better than cells lacking Isc1 (58). Together, these studies demonstrate that Isc1 plays an important role in mitochondrial function during the diauxic shift. Future studies should reveal the functional role of phytoceramide in mitochondria. In log-phase cells, Isc1 is responsible for part of the increase in ceramide that occurs when yeast are heat shocked (54, 59, 60), and it also plays an uncharacterized role in sporulation (60). A new role for Isc1 in heat stress and the regulation of sphingolipid synthesis is presented in the next section.
A seminal advance in our understanding of how cells regulate de novo ceramide synthesis and, thus, the synthesis of complex sphingolipids was made recently by Powers and colleagues (61), who showed that the Target Of Rapamycin Complex 2 (TORC2) controls the activity of ceramide synthase. TOR is a conserved protein kinase that senses nutrients and stresses and coordinates metabolism both temporarily and spatially to control cell growth (62). All known eukaryotes contain two TOR protein complexes, TORC1, which is inhibited by rapamycin, a bacterial macrocyclic lactone, and TORC2, which is rapamycin-insensitive.
A connection between sphingolipids and the TORs was first suggested by the work of Beeler et al. (49), who were studying calcium homeostasis in yeast. They isolated a mutation in the CSG2 gene that conferred sensitivity to 100 mM Ca2+. A screen for temperature-sensitive mutations that bypassed the calcium sensitivity of csg2 cells identified several genes, including TOR2 and AVO3/TSC11, that encode components of TORC2 (24). TORC2 controls the organization of the actin cytoskeleton in yeast and mammals (62).
To elucidate the connection between TOR signaling and sphingolipids, Aronova et al. (61) isolated an allele of AVO3 (avo3-30) that diminished growth at 30°C. Temperature was a concern because previous alleles required higher temperatures to impair growth and high temperatures enhance DHS, PHS, and ceramide levels (12), which might mask the effect of an avo3 mutation on ceramide synthesis. At 25°C avo3-30 cells grew like wild-type cells, but growth slowed within 5–6 h after a shift to 30°C. Aronova et al. (61) found that 3 h after shifting avo3-30 cells to 30°C, the concentration of the major yeast ceramide species containing PHS and a C26 fatty acid was reduced by 5-fold compared with wild-type cells. The level of minor ceramide species having shorter fatty acid chains was reduced by 10-fold. Just as telling, the concentration of the major ceramides was reduced by 2-fold even in avo3-30 cells grown at 25°C. These results predict that avo3-30 cells have reduced ceramide synthase activity, a prediction that was confirmed by measuring enzyme activity in microsomes isolated from cells grown at 30°C.
Aronova et al. (61) next examined how TORC2s affect ceramide synthase activity. They focused on the Ypk2 protein kinase because it is activated by TORC2 (63) and because mutant ypk2 and avo3-30 cells show similar defects in cell wall integrity and actin polarization. Insightful data were obtained using a constitutively active allele of YPK2: all avo3-30 phenotypes including the ceramide deficiency were reversed. These results argue that Ypk2 acts downstream of TORC2 to activate ceramide synthase activity (Fig. 2 ). The actual molecular mechanism will require further work to determine whether Ypk2 directly phosphorylates a subunit of ceramide synthase to govern enzyme activity or whether some other Ypk2 substrate regulates the activity.
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Previous work had shown that blocking steps in sphingolipid synthesis downstream of DHS and PHS causes their accumulation (25, 26, 64), as does heat stress (59, 65). Aronova et al. (61) found that avo3-30 cells do indeed accumulate these LCBs, as expected for cells with reduced ceramide synthase activity. DHS and PHS are known to activate the Pkh1 and Pkh2 protein kinases, which, along with TORC2, activate Ypk2 (reviewed in Ref. 12). There is also an indication that DHS and PHS can act directly to partially activate Ypk2 (66). Thus, Aronova et al. (61) speculated that DHS and PHS act in a feed-forward manner to coregulate Ypk2 along with TORC2, thereby controlling ceramide synthase activity and the flux of LCBs that are incorporated into ceramides and complex sphingolipids (Fig. 2).
These studies in yeast begin to reveal how cells promote ceramide and sphingolipid synthesis when conditions favor growth and how they reduce synthesis when stresses threaten cells and impede growth. Because the early steps in de novo sphingolipid synthesis are reasonably conserved, mammalian TORC2 may play a role in regulating de novo ceramide synthesis. However, mammals have at least six ceramide synthases that make ceramides with fatty acids of a particular chain length (67). These enzymes also display unique temporal and cell-specific expression patterns. Given this diversity, it is unlikely that mTORC2 regulates all of these enzymes, but it could govern some in certain cell types. The mammalian homolog of Ypk2 is the serum- and glucocorticoid-inducible protein kinase, which may transmit the signal from mTORC2 to some ceramide synthases. But it would not be surprising if other members of the AGC kinase family, including Akts/PKBs, transmitted the mTORC2 signal to ceramide synthases, because Akt/PKB functions downstream of mTORC2 (62). Mammalian ceramide synthases are already receiving much attention, but the yeast results should stimulate a more focused interest and may pave the way to understanding how de novo ceramide synthesis is regulated in mammals. Such understanding could lead to better chemotherapeutic drugs, some of which promote killing by enhancing de novo ceramide synthesis in ways that are not understood (68, 69).
It has been more than 10 years since heat stress was observed to induce an increase in LCBs (59, 65), but the molecular mechanism(s) underlying the increase has remained unclear. The increases are substantial, but transient. For example, C18-DHS and C18-PHS increase by 2- to 3-fold and C20-DHS and C20-PHS increase by >100-fold, with the peaks appearing at 5–10 min after the temperature shift. Thereafter, LCBs return to basal values, even though the cells remain at an increased temperature. Cowart and Hannun (70) have presented evidence that the transient increase in LCBs is at least partly driven by an increased uptake of serine from the culture medium and is not attributable to changes in the specific activity of SPT (Fig. 1). However, heat-induced increases in C20-DHS and C20-PHS were reduced by 75% in cells lacking the Tsc3 subunit of SPT, indicating that this protein, which is only essential for growth at increased temperatures (21), plays a role in the synthesis of C20-LCBs. Previous studies with mammalian cells indicated that substrate availability played a role in determining the rate of LCB production (71, 72). The availability of the two SPT substrates, fatty acyl-CoAs and serine, was examined for effects on LCB synthesis, and serine was found to be important (70). Using a mutant strain defective in serine synthesis (ser3ser33), serine taken up from the culture medium was shown to be responsible for the heat-induced increase in LCBs. In addition, the rate of serine uptake was shown to be stimulated by heat. Two other factors besides heat that are known to increase amino acid uptake, the acidification of the culture medium and the addition of glucose to glucose-starved cells, also increased LCB production. Thus, the rate of de novo LCB synthesis in yeast is at least partially controlled by the rate of serine uptake.
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CELLULAR PROCESSES REGULATED BY LCBS |
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TOPABSTRACTINTRODUCTIONSPHINGOLIPID METABOLISMCELLULAR PROCESSES REGULATED BY...SECRETORY PATHWAY, ENDOCYTOSIS,...SUMMARY AND FUTURE DIRECTIONSREFERENCES |
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Insight into the signal transduction pathways regulated by LCBs was found first while attempting to identify genes whose overexpression bypassed the growth inhibition caused by myriocin (73). Myriocin inhibits SPT and blocks cell growth by curtailing sphingolipid synthesis (74). Sun et al. (73) found that the YPK1 gene, when present on a multicopy vector, bypassed the myriocin block. Ypk1 is a protein kinase that plays a role in cell wall maintenance and actin cytoskeleton dynamics (75, 76), endocytosis (77), and translation during nitrogen starvation and nutrient sensing (78). Ypk1 and its paralog Ypk2 are structural and functional homologs of mammalian serum- and glucocorticoid-inducible kinase (79).
Because the protein kinase Pkh1 was known to phosphorylate and activate Ypk1 (79), multiple copies of PKH1 were examined and found to also bypass the growth inhibition caused by myriocin (73). These results suggested that some sphingolipid activated the Pkh1/Ypk1-Ypk2 signaling pathway. Pkh1 is a homolog of mammalian phosphoinositide-dependent protein kinase 1 (PDK1), which is well known for being activated by binding to 3-phosphoinositides via a pleckstrin homology domain. However, Pkh1 has no pleckstrin homology domains and was not activated by phosphoinositides in vitro (79). Sun et al. (73) showed that myriocin-treated cells lacked a phosphorylated and presumably active form of Ypk1 and that this form reappeared in vivo when PHS was added to the culture medium, even with myriocin still present. These experiments supported, but did not prove, the hypothesis that PHS activated Pkh1 or its homolog Pkh2, which then phosphorylated and activated Ypk1. After these initial experiments, many laboratories contributed data supporting the hypothesis that LCBs act to activate Pkh1/2, which then phosphorylate and contribute to the activation of kinases, including Ypk1/2, Pkc1, and Sch9 (Fig. 3
). Contributions made by individual laboratories to our understanding of the LCB
Pkh1/2 pathway have been described in detail in previous reviews (11, 12).
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Previous results (24) implied a connection or connections between the LCBPkh1/2 and TOR signaling pathways, but the mechanism was unclear. This void in our knowledge is beginning to be filled with the realization that these two pathways, along with the calcineurin signaling pathway, modulate the phosphorylation/dephosphorylation cycle of the Slm1 and Slm2 proteins during heat stress to control actin polarization, endocytosis, and sphingolipid metabolism (82–84).
SLM1 and SLM2 have overlapping functions, and at least one of them is required for viability (85). A distinguishing feature of the Slm proteins is a pleckstrin homology domain that enables them to bind phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is made transiently, for example during heat shock, on the inner leaflet of the plasma membrane, where it serves to initially anchor the Slm proteins (85, 86). The strength of Slm binding to the membrane is strengthened by interactions with the Avo2 and Bit61 subunits of TORC2 (85, 87), and this interaction also facilitates the phosphorylation of Slm1 and Slm2 by TORC2 (85). Dual binding enables the Slm proteins to mediate effects downstream of both the PIP2 and TORC2 signaling pathways that control roles of the actin cytoskeleton essential for growth, cell wall integrity, and receptor-mediated endocytosis.
Heat stress was recently shown to initiate the dephosphorylation and rephosphorylation of the Slm proteins (83–85). Dephosphorylation was shown to be mediated by the protein phosphatase calcineurin, which binds to the Slm proteins (82–84) and plays roles in a variety of stress responses. Because heat also generates a transient increase in PHS and DHS, which then activate the Pkh1/2 signaling pathway (Fig. 3), it was hypothesized that PHS played a role in the phosphorylation of the Slm proteins. Several types of experiments support this hypothesis, but the most physiologically significant used myriocin to reduce the concentration of PHS and DHS and demonstrate a reduction in the heat-induced phosphorylation of Slm1 and Slm2 (83, 84). The idea that the Slm proteins depend upon sphingolipid signaling or metabolism or both to carry out their essential role in cell growth was supported also by the observation that deletion of either slm1 or slm2 made cells more sensitive to myriocin (83, 84). Furthermore, combining a slm1 mutation with a nonessential mutation in a sphingolipid metabolic gene, including fen1, lcb4, or csg2, generated cells that were more sensitive to myriocin than the single mutants. Likewise, the actin cytoskeleton was more disrupted in the double mutant than in the single mutants, supporting the idea that Slm function depends upon sphingolipids (84). Finally, phosphorylation of Slm1 has been shown to depend upon the Pkh kinases. In cells carrying a temperature-sensitive allele of pkh1ts and having pkh2 deleted, the basal and heat-induced level of phosphorylated Slm was reduced (84). This phosphorylation seems to be independent of TORC2.
Attempts to determine whether Pkh1 directly phosphorylates Slm1 and Slm2 suggest that they do not, because His6-Pkh1 produced in and purified from Escherichia coli failed to phosphorylate purified GST-Slm1 or GST-Slm2 in vitro. However, these experiments are inconclusive, because Pkh1 made in E. coli may not be properly activated or the in vitro reaction conditions may not be suitable for phosphorylation of the Slm proteins. Whether or not the Slm proteins are phosphorylated by one of the kinases that acts downstream of Pkh1/2 is also unresolved. Serine 659 appears to be the site phosphorylated by the Pkh1/2 pathway, and this phosphorylated residue is essential for Slm1 to support growth at higher but not at lower temperatures (84). Although the biochemical function or activity of Slm1 and Slm2 is unknown, it seems likely that they function downstream of the LCBPkh1/2 pathway to control the polarization of the actin cytoskeleton. This conclusion is based upon a mutant strain having a negatively charged aspartic acid residue in place of serine 659. The negative charge mimics phosphorylation and produces constitutive Slm1 activity, which, when overexpressed, is able to restore actin polarization at increased temperatures in the pkh1ts pkh2 mutant strain (84). However, the SLM1S659D allele does not restore the growth of pkh1ts pkh2 cells at increased temperatures, indicating that Slm1 does not mediate all essential Pkh1/2 functions. Understanding the biochemical action of the Slm proteins would provide an important advance in understanding how the LCBPkh1/2 pathway regulates the actin cytoskeleton.
The PIP2 pathway was recently found to regulate the synthesis of IPC via the Slm1 and Slm2 proteins, which downregulate the activity of Isc1 (Fig. 1), the enzyme that cleaves polar head groups from complex sphingolipids (82). Previous studies had implicated the PIP2 pathway in regulating IPC levels, but the mechanism was unclear. PIP2 is synthesized from phosphatidylinositol by the sequential action of Stt4, a phosphatidylinositol 4-kinase that makes phosphatidylinositol 4-phosphate (PI4P), which is then converted to PIP2 by the Mss4 enzyme. Previous data had implied a connection between the PIP2 pathway and sphingolipids. A screen for mutations that could bypass the calcium sensitivity of csg2 cells identified mss4 (24). In addition, the screen for genes that bypassed growth inhibition by myriocin also uncovered MSS4 as a multicopy suppressor gene in addition to identifying YPK1 (88). In recently reported studies, synthetic genetic array analysis was used to search for genes that regulate or work downstream of PI4P generated by Stt4. Strains in the nonessential yeast deletion collection were combined with a temperature-sensitive stt4ts strain to identify double mutants with slow or impaired growth (82). This screen identified FEN1 and SUR4, required for the synthesis of C26 fatty acids that are a necessary component of sphingolipids (Fig. 1), and thereby implicated sphingolipids as targets of the PIP2 pathway.
The synthetic genetic array results were followed up by several types of experiments, and the key results will be discussed here. Analyses of mutants that bypassed the calcium sensitivity of csg2 suggested that they restored growth by reducing the level of a species of IPC termed IPC-C. Therefore, the levels of this and other complex sphingolipids plus ceramide were analyzed by radiolabeling cells grown at 26°C and 38°C with [3H]serine and measuring radioactive sphingolipids by thin-layer chromatography and autoradiography (82). At 26°C, the levels of IPCs, MIPC, and ceramide were reduced in stt4ts cells compared with wild-type cells, and the difference was even greater in cells grown at 38°C. Similar results were observed with mss4ts cells. These and other data confirm the hypothesis that Stt4 and Mss4 regulate IPC levels, and they do so independently of their known role in the activation of the Rho1/Pkc1 cell wall integrity pathway (reviewed in Ref. 89).
Because Slm1 and Slm2 operate downstream of the PIP2 and TORC2 pathways, they were examined for effects on IPC synthesis. IPC-C and other complex sphingolipids were less abundant in slm1ts slm2 cells grown at 26°C or 38°C, similar to what was found in stt4ts and mss4ts cells. The connection between the Slm proteins and sphingolipids was further strengthened by analyzing the effect of slm1 and slm2 mutations on csg2 cells. Interestingly, slm1ts slm2 csg2 cells did not grow, which implies a functional relation between the Slm protein and Csg2. By deleting one or the other SLM gene, it was observed that slm1csg2 cells grew at 26°C but not 38°C and slm2csg2 cells grew at both temperatures. The difference between these two types of double mutants is probably a reflection of Slm1 being more abundant than Slm2. These data suggest a functional interaction between Slm1 and Csg2. This idea is strengthened by the finding that the actin cytoskeleton is completely depolarized in slm1csg2 cells grown at 38°C, whereas the single mutants show no defect.
The relationship between Slm1 and Csg2 was explored further by assessing the phosphorylation of Slm1 during heat stress. Although Slm1 phosphorylation increased during a 60 min heat shock in wild-type cells, there was almost no increase in csg2 cells. Because phosphorylation is mediated by TORC2, one interpretation of these data is that TORC2 function is disrupted by heat stress in csg2 cells. However, another interpretation is that a protein phosphatase is activated when csg2 is deleted. Because calcineurin subunits had been found to interact with Slm2 and both Slm2 and Slm1 have putative calcineurin binding motifs, the phosphorylation of Slm1 was examined in csg2 cells treated with the calcineurin inhibitor FK506. Phosphorylation of Slm1 was partially restored in FK506-treated cells, consistent with calcineurin being responsible for dephosphorylation in csg2 cells. Measurement of calcineurin activity showed that it was increased by 3-fold in csg2 cells, increased slightly in slm1 cells, and increased by 10-fold in slm1csg2 double mutant cells. Likewise, it was increased in mss4ts and tor2ts cells. Together, these data indicate that Slm1, Slm2, and Csg2 cooperate to downregulate calcineurin activity (Fig. 4
). Furthermore, they imply the regulation of IPC metabolism by calcineurin, and analysis of sphingolipids showed that activating calcineurin by adding 100 mM Ca2+ to cells accelerated the conversion of IPC to MIPC.
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cells. Also, deletion of ISC1, similar to deletion of calcineurin, restored IPC levels in slm1ts slm2 cells to wild-type levels and reversed the ts phenotype. Moreover, deletion of ISC1 rescued the nonviable phenotype of slm1csg2 cells. These data imply that misregulation of the hyperactivation of Isc1 and calcineurin most likely explains the phenotypes associated with the loss of Slm1 and Slm2 activity. In fact, inactivation of both ISC1 and calcineurin rescued the actin and viability defects seen in slm1ts slm2 cells at the restrictive temperature. The data of Tabuchi et al. (82) are summarized in the model diagrammed in Fig. 4. They propose that the Slm proteins function downstream of the PIP2 and TORC2 signaling pathways to enable cells to respond to stresses. Part of the response involves recruitment of the Slms to the plasma membrane by binding to PIP2, followed by phosphorylation and activation mediated by TORC2. Activated Slms then downregulate Isc1 activity and control the breakdown of complex sphingolipids, especially IPC-C. The Slms also downregulate calcineurin phosphatase activity, which interacts with Csg2 in an unknown manner to regulate the conversion of IPC-C to MIPC. Dephosphorylation of the Slm proteins by calcineurin acts as a negative feedback loop to modulate Slm function. In this context, the Slm proteins serve to modulate changes in membrane composition and/or architecture in response to stresses. Other results from Tabuchi et al. (82) demonstrate the PIP2 control of the Rho1/Pkc1 pathway independent of its control of sphingolipid metabolism. Finally, it is likely that IPC, possibly a specific pool of IPC-C, regulates actin organization and viability. Identifying this pool of IPC-C could provide important clues for understanding how such regulation occurs.
Overall, the realization that the PIP2 and TORC2 pathways use the Slm proteins and calcineurin to regulate sphingolipid metabolism provides a framework for a detailed mechanistic understanding of how sphingolipid synthesis and breakdown are integrated with the need for cells to grow and to be able to interrupt growth to respond to life-threatening stresses.
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SECRETORY PATHWAY, ENDOCYTOSIS, AND THE PLASMA MEMBRANE |
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TOPABSTRACTINTRODUCTIONSPHINGOLIPID METABOLISMCELLULAR PROCESSES REGULATED BY...SECRETORY PATHWAY, ENDOCYTOSIS,...SUMMARY AND FUTURE DIRECTIONSREFERENCES |
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These new studies used strains carrying the semidominant SLC1-1 mutation, which carries a Q44L substitution in the Slc1 protein, a 1-acyl-sn-glycerol-3-phosphate acyltransferase (17). This mutation enables strains carrying lcb1, which is normally lethal because cells cannot make LCBs, to survive by catalyzing the incorporation of C26 fatty acids into the sn-2 position of glycerolipids, which then mimic yeast ceramides and serve as substrates for the enzymes that add the polar head groups found in yeast sphingolipids (Fig. 1). Thus, because of the lcb1 mutation, SLC1-1 cells lack LCBs and ceramides but they contain a set of novel glycerol-based lipids with the polar head groups and C26 fatty acid found in yeast sphingolipids (16). When SLC1-1 cells are fed LCBs, they make sphingolipids and behave like wild-type cells.
Sphingolipids and sterols associate with each other in membranes to form microdomains or raft-like structures (93, 94) with high affinity for specific membrane proteins, including Pma1 (95). The new work by Gaigg, Toulmay, and Schneiter (90) shows that the C26-containing inositol glycerophospholipids in SLC1-1 cells can functionally substitute for sphingolipids in forming detergent-resistant membrane domains containing Pma1 and another raft-associated protein, Gas1. Gaigg, Toulmay, and Schneiter (90) also demonstrated that the C26-containing inositol glycerophospholipids in SLC1-1 cells substitute for the functions of sphingolipids in the secretory pathway and facilitate the normal delivery of Pma1 to the plasma membrane. The essential function(s) of the C26 acyl chain in the suppressor lipids was demonstrated by introducing a sur4 (elo3) mutation into the SLC1-1 strain. Sur4 is the Elop necessary for elongating fatty acids up to 26 carbon atoms in length (Fig. 1). sur4 cells make C22-containing sphingolipids and are viable (35), but SLC1-1 lcb1sur4 cells are not viable unless they are fed an LCB so that they can make sphingolipids. These experiments establish that the C26 acyl group in suppressor lipids is essential and that two of its essential roles are to direct Pma1 to the plasma membrane and to stabilize it in a detergent-resistant membrane environment (90).
Gaigg, Toulmay, and Schneiter (90) also examined endocytosis in SLC1-1 lcb1 cells that lack sphingolipids, because the stability of Pma1 in the plasma membrane of these cells could simply be attributable to a block in endocytosis. However, this seems not to be the case, because uptake of the fluorescent dye Lucifer Yellow, a marker for fluid-phase endocytosis, was similar in SLC1-1 lcb1 cells that contained or lacked sphingolipids and in wild-type cells. These results create a conundrum, because previous data showed that LCBs are essential for endocytosis (96, 97). Gaigg, Toulmay, and Schneiter (90) suggest that the protein phosphatase PP2A, a negative regulator of endocytosis (98), may be less active in SLC1-1 lcb1 cells because they lack ceramide, which is thought to be an activator of PP2A (99, 100). This explanation probably represents only part of a very complex picture, because the absence of sphingolipids in SLC1-1 lcb1 cells likely increases some and decreases other regulatory pathways to distort cellular processes.
Results from another recent study (30) exemplify how yeast cells can adapt to changes in their sphingolipids and still remain viable. In that study, a strain lacking the yeast ceramide synthases Lag1 and Lac1 (Fig. 1) and expressing the mammalian ceramide synthase Lass5 made sphingolipids containing primarily DHS, rather than the usual PHS, and C16 and C18 fatty acids rather than the usual C24 and C26 fatty acids. Although 97% of the sphingolipids in these cells lacked C24 or C26 fatty acids, these VLCFAs were still essential, because a block in the fatty acid elongation cycle produced by deleting TSC13 (Fig. 1) was lethal. The authors suggest that the VLCFAs may be necessary for the synthesis of glycerolipids with a VLCFA in the sn-1 position (101).
Another plasma membrane protein, the general amino acid permease Gap1, depends in various ways upon sphingolipids (102). Gap1 synthesized in lcb1-100 cells at a restrictive temperature is transported to the plasma membrane but is not incorporated into detergent-resistant lipid rafts, lacks amino acid transport activity, and is rapidly endocytosed in a ubiquitin-mediated manner. The authors conclude that these phenotypes result from Gap1 synthesis in the ER in the absence of sphingolipids and that sphingolipids provide a microenvironment for producing Gap1 with a conformation necessary for transport activity, the association with lipid rafts, and normal stability.
The Slm proteins mentioned above have also been shown to play roles in endocytosis. Sphingolipids are normally involved in regulating the turnover and endocytosis of the uracil permease Fur4 during heat stress (97, 103–105). However, in cells lacking Slm protein activity, Fur4 was quite stable during heat shock and accumulated in a ubiquitinated form that would normally be a substrate for endocytosis but that fails to be endocytosed in slm1ts slm2 cells (83). Thus, the Slm proteins are required for a step in the endocytosis of Fur4 that follows ubiquitylation. The Slm proteins, however, are not required generally for endocytosis, because slm mutant cells do endocytose Lucifer Yellow at high temperatures (83).
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TOPABSTRACTINTRODUCTIONSPHINGOLIPID METABOLISMCELLULAR PROCESSES REGULATED BY...SECRETORY PATHWAY, ENDOCYTOSIS,...SUMMARY AND FUTURE DIRECTIONSREFERENCES |
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Finally, nearly all of our current understanding of sphingolipid metabolism and functions comes from studies done with rapidly dividing cells growing in log phase and with glucose as the carbon source. Sphingolipids are likely to play novel roles under different growth conditions and in different parts of their life cycle, including stationary phase, spore formation, and germination, and in mature spores.
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ACKNOWLEDGMENTS |
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Manuscript received January 31, 2008 and in revised form February 20, 2008.
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TOPABSTRACTINTRODUCTIONSPHINGOLIPID METABOLISMCELLULAR PROCESSES REGULATED BY...SECRETORY PATHWAY, ENDOCYTOSIS,...SUMMARY AND FUTURE DIRECTIONSREFERENCES |
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