NORMAL GENOMIC

• Kohlwein SD et al.
• Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae.
• Mol Cell Biol. 2001; 21: 109-25
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• The TSC13/YDL015c gene was identified in a screen for suppressors of the calcium sensitivity of csg2Delta mutants that are defective in sphingolipid synthesis. The fatty acid moiety of sphingolipids in Saccharomyces cerevisiae is a very long chain fatty acid (VLCFA) that is synthesized by a microsomal enzyme system that lengthens the palmitate produced by cytosolic fatty acid synthase by two carbon units in each cycle of elongation. The TSC13 gene encodes a protein required for elongation, possibly the enoyl reductase that catalyzes the last step in each cycle of elongation. The tsc13 mutant accumulates high levels of long-chain bases as well as ceramides that harbor fatty acids with chain lengths shorter than 26 carbons. These phenotypes are exacerbated by the deletion of either the ELO2 or ELO3 gene, both of which have previously been shown to be required for VLCFA synthesis. Compromising the synthesis of malonyl coenzyme A (malonyl-CoA) by inactivating acetyl-CoA carboxylase in a tsc13 mutant is lethal, further supporting a role of Tsc13p in VLCFA synthesis. Tsc13p coimmunoprecipitates with Elo2p and Elo3p, suggesting that the elongating proteins are organized in a complex. Tsc13p localizes to the endoplasmic reticulum and is highly enriched in a novel structure marking nuclear-vacuolar junctions.

• Bagnat M, Keranen S, Shevchenko A, Shevchenko A, Simons K
• Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast.
• Proc Natl Acad Sci U S A. 2000; 97: 3254-9
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• Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.

• Hettema EH, Girzalsky W, van Den Berg M, Erdmann R, Distel B
• Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins.
• EMBO J. 2000; 19: 223-33
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• The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Delta and pex19Delta, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexDelta mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.

• Springer S et al.
• The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae.
• Proc Natl Acad Sci U S A. 2000; 97: 4034-9
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• To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of S. cerevisiae (p24Delta8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p). The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells. We find that p24Delta8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single Deltaemp24 deletion strain. Thus, p24 proteins do not have an essential function in the secretory pathway. Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles.

• Trilla JA, Duran A, Roncero C
• Chs7p, a new protein involved in the control of protein export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin synthesis in Saccharomyces cerevisiae.
• J Cell Biol. 1999; 145: 1153-63
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• The Saccharomyces cerevisiae CHS7 gene encodes an integral membrane protein located in the ER which is directly involved in chitin synthesis through the regulation of chitin synthase III (CSIII) activity. In the absence of CHS7 product, Chs3p, but not other secreted proteins, is retained in the ER, leading to a severe defect in CSIII activity and consequently, to a reduced rate of chitin synthesis. In addition, chs7 null mutants show the yeast phenotypes associated with a lack of chitin: reduced mating efficiency and lack of the chitosan ascospore layer, clear indications of Chs7p function throughout the S. cerevisiae biological cycle. CHS3 overexpression does not lead to increased levels of CSIII because the Chs3p excess is retained in the ER. However, joint overexpression of CHS3 and CHS7 increases the export of Chs3p from the ER and this is accompanied by a concomitant increase in CSIII activity, indicating that the amount of Chs7p is a limiting factor for CSIII activity. Accordingly, CHS7 transcription is increased when elevated amounts of chitin synthesis are detected. These results show that Chs7p forms part of a new mechanism specifically involved in Chs3p export from the ER and consequently, in the regulation of CSIII activity.

• Barz WP, Walter P
• Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.
• Mol Biol Cell. 1999; 10: 1043-59
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• Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

• Dickson RC
• Sphingolipid functions in Saccharomyces cerevisiae: comparison to mammals.
• Annu Rev Biochem. 1998; 67: 27-48
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• Many roles for sphingolipids have been identified in mammals. Available data suggest that sphingolipids and their intermediates also have diverse roles in Saccharomyces cerevisiae. These roles include signal transduction during the heat stress response, regulation of calcium homeostasis or components in calcium-mediated signaling pathways, regulation of the cell cycle, and functions as components in trafficking of secretory vesicles from the endoplasmic reticulum to the Golgi apparatus and as the lipid moiety in many glycosylphosphatidylinositol-anchored proteins. S. cerevisiae is likely to be the first organism in which all genes involved in sphingolipid metabolism are identified. This information will provide an unprecedented opportunity to determine, for the first time in any organism, how sphingolipid synthesis is regulated. Through the use of both genetic and biochemical techniques, the identification of the complete array of processes regulated by sphingolipid signals is likely to be possible, as is the quantification of the physiological contribution of each.

• David D, Sundarababu S, Gerst JE
• Involvement of long chain fatty acid elongation in the trafficking of secretory vesicles in yeast.
• J Cell Biol. 1998; 143: 1167-82
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• Members of the synaptobrevin/VAMP family of v-SNAREs are thought to be essential for vesicle docking and exocytosis in both lower and higher eukaryotes. Here, we describe yeast mutants that appear to bypass the known v-SNARE requirement in secretion. Recessive mutations in either VBM1 or VBM2, which encode related ER-localized membrane proteins, allow yeast to grow normally and secrete in the absence of Snc v-SNAREs. These mutants show selective alterations in protein transport, resulting in the differential trafficking and secretion of certain protein cargo. Yet, processing of the vacuolar marker, carboxypeptidase Y, and the secreted protein, invertase, appear normal in these mutants indicating that general protein trafficking early in the pathway is unaffected. Interestingly, VBM1 and VBM2 are allelic to ELO3 and ELO2, two genes that have been shown recently to mediate the elongation of very long chain fatty acids and subsequent ceramide and inositol sphingolipid synthesis. Thus, the v-SNARE requirement in constitutive exocytosis is abrogated by mutations in early components of the secretory pathway that act at the level of lipid synthesis to affect the ability of secretory vesicles to sort and deliver protein cargo.

• Pilon M, Schekman R, Romisch K
• Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.
• EMBO J. 1997; 16: 4540-8
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• Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.

• Skrzypek M, Lester RL, Dickson RC
• Suppressor gene analysis reveals an essential role for sphingolipids in transport of glycosylphosphatidylinositol-anchored proteins in Saccharomyces cerevisiae.
• J Bacteriol. 1997; 179: 1513-20
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• Sphingolipids are normally necessary for growth of Saccharomyces cerevisiae cells, but mutant strains that bypass the need for sphingolipids have been identified. Such bypass mutants fail to grow under stressful conditions, including low pH (pH 4.1), when they lack sphingolipids. To begin to understand why sphingolipids seem to be necessary for coping with low-pH stress, we screened a genomic library and selected a suppressor gene, CWP2 (cell wall protein 2), that when present in multiple copies partially compensates for the lack of sphingolipids and enhances survival at low pH. To explain these results, we present evidence that sphingolipids are required for a normal rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins, including Cwp2 and Gas1/Gpg1, from the endoplasmic reticulum (ER) to the Golgi apparatus. The effect of sphingolipids is specific for transport of GPI-anchored proteins because no effect on the rate of transport of carboxypeptidase Y, a non-GPI-anchored protein, was observed. Since the Gasl protein accumulated in the ER with a GPI anchor in cells lacking sphingolipids, we conclude that sphingolipids are not necessary for anchor attachment. Therefore, sphingolipids must be necessary for a step in formation of COPII vesicles or for their transport to the Golgi apparatus. Our data identify the Cwp2 protein as a vital component in protecting cells from the stress of low pH.

• Cox JS, Chapman RE, Walter P
• The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.
• Mol Biol Cell. 1997; 8: 1805-14
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• The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

• Knop M, Finger A, Braun T, Hellmuth K, Wolf DH
• Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.
• EMBO J. 1996; 15: 753-63
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• The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called 'der' for 'degradation in the ER'. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.

• Wu WI et al.
• Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1.
• J Biol Chem. 1995; 270: 13171-8
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• The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined. Fumonisin B1 inhibited the growth of yeast cells. Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner. The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells. Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1. Fumonisin B1 inhibited the synthesis of the inositol-containing sphingolipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids. The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined. Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined. However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase. These are key enzymes responsible for the synthesis of lipids in yeast. The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases.

• Riezman H, Horvath A, Manning-Krieg U, Movva R
• Intracellular transport of GPI-anchored proteins by yeast.
• Braz J Med Biol Res. 1994; 27: 323-6
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• We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas1p. The need for ceramide can be overcome in the mutant strain.

• Horvath A, Sutterlin C, Manning-Krieg U, Movva NR, Riezman H
• Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.
• EMBO J. 1994; 13: 3687-95
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• Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide-containing, mild base-resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI-anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI-anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI-anchored proteins.