SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32- kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH- terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.     

The yeast SEC53 gene encodes phosphomannomutase

Yeast sec53 cells incubated at a restrictive temperature (37 degrees C) accumulate inactive and incompletely glycosylated forms of secretory proteins within the lumen of the endoplasmic reticulum. A defect in glycosylation of alpha-factor precursor has been reproduced in vitro using membranes and cytosol isolated from sec53 mutant cells. Normal glycosylation is restored in reactions supplemented with a cytosolic fraction from wild type cells, with GDP-mannose, or with mannose 1-phosphate and GTP, but not with mannose 6-phosphate and GTP. This pattern of stimulation suggests that extracts of sec53 cells are deficient in phosphomannomutase activity or in the production of a precursor of mannose 1-phosphate. Several lines of evidence demonstrate that SEC53 encodes the yeast phosphomannomutase. Direct assay of soluble fractions from independent alleles of sec53 shows low to negligible phosphomannomutase, but nearly normal levels of phosphomannoisomerase activity. The residual phosphomannomutase activity in mutant cell lysates is thermolabile in proportion to the severity of the sec53 cell growth defect. Introduction of the SEC53 gene on a multicopy plasmid into sec53 or wild type yeast and into Salmonella typhimurium results in an increase in phosphomannomutase activity that correlates with elevated expression of the Sec53 protein. Finally, the Sec53 protein and phosphomannomutase activity cofractionate exactly in a 70-fold partial purification involving gel filtration and DEAE chromatography. The secretory defect in sec53 cells may now be explained by a deficit in GDP-mannose production.     

Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.     

Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.     

SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast

Three new and likely related components of the cellular fusion machinery have been purified from bovine brain cytosol, termed alpha-SNAP (35 kd), beta-SNAP (36 kd), and gamma-SNAP (39 kd). Transport between cisternae of the Golgi complex measured in vitro requires SNAP activity during the membrane fusion stage, and each SNAP is capable of binding the general cellular fusion protein NSF to Golgi membranes. The SNAP-NSF-membrane complex may be an early stage in the assembly of a proposed multisubunit "fusion machine" on the target membrane. SNAP transport factor activity is also found in yeast. Yeast cytosol prepared from a secretion mutant defective in export from the endoplasmic reticulum (sec17) lacks SNAP activity, which can be restored in vitro by the addition of pure alpha-SNAP, but not beta- or gamma-SNAPs. These data suggest that the mechanism of action of SNAPs in membrane fusion is conserved in evolution.     

Prepro-alpha-factor has a cleavable signal sequence

MAT alpha Saccharomyces cerevisiae secrete a small peptide mating pheromone termed alpha-factor. Its precursor, prepro-alpha-factor, is translocated into the endoplasmic reticulum and glycosylated at three sites. The glycosylated form is the major product in a yeast in vitro translation/translocation system. However, there is another translocated, nonglycosylated product that contains a previously unidentified modification. Contrary to previous results suggesting that the signal sequence of prepro-alpha-factor is not cleaved, amino-terminal radiosequencing has identified this product as prepro-alpha-factor without its signal sequence, that is, pro-alpha-factor. The translocated, glycosylated proteins are also processed by signal peptidase. Moreover, we have found that both purified eukaryotic and prokaryotic signal peptidase can process prepro-alpha-factor. Experiments using a yeast secretory mutant (sec 18) blocked in transport from the endoplasmic reticulum to the Golgi indicate that the protein is also cleaved in vivo. Finally, characterization of the Asn-linked oligosaccharide present on pro-alpha-factor in the yeast in vitro system by use of specific glucosidase and mannosidase inhibitors indicates that they have had the three terminal glucoses and probably one mannose removed. Therefore they most likely consist of Man8GlcNAc2 structures, identical to those found in the endoplasmic reticulum in vivo.     

Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae.

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.     

The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.     

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.     

Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro.

The SEC23 gene product (Sec23p) is required for transport of secretory, plasma membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex in Saccharomyces cerevisiae. Molecular cloning and biochemical characterization demonstrate that Sec23p is an 84 kd unglycosylated protein that resides on the cytoplasmic surface of a large structure, possibly membrane or cytoskeleton. Vigorous homogenization of yeast cells or treatment of yeast lysates with reagents that desorb peripheral membrane proteins releases Sec23p in a soluble form. Protein transport from the endoplasmic reticulum to the Golgi in vitro depends upon active Sec23p. Thermosensitive transport in sec23 mutant lysates is restored to normal when a soluble form of wild-type Sec23p is added, providing a biochemical complementation assay for Sec23p function. Gel filtration of yeast cytosol indicates that functional Sec23p is a large oligomer or part of a multicomponent complex.     

Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.     

O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation

Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pmt2p). O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane. Deletion of PMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.     

Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation

The endoplasmic reticulum contains a protein quality control system that discovers malfolded or unassembled secretory proteins and subjects them to degradation in the cytosol. This requires retrograde transport of the respective proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon. In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and degradation. Ubiquitination of mutated and malfolded proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p. In addition, several new membrane components of the endoplasmic reticulum are required for degradation. Here we present the topology of the previously discovered RING-H2 finger protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane. The protein spans the membrane six times. The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm. Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the endoplasmic reticulum-associated protein degradation process.     

Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro- alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.     

Secretion of Saccharomyces cerevisiae Killer Toxin: Processing of the Glycosylated Precursor

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2. Protoxin stability in kex1 kex2 double mutants indicated the order kex1 --> kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1- and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.     

In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.

Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp- Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro-alpha-factor (MF alpha 1) N-terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature-sensitivity of growth and protein translocation. At the non-permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro-carboxypeptidase Y that had been accumulated in the cytosol at the non-permissive temperature could be post-translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post-translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature-sensitive component is membrane-associated in ptl1, and that the membranes derived from this strain show a reversible temperature-sensitive translocation phenotype in vitro.     

Inhibition of GTP hydrolysis by Sar1p causes accumulation of vesicles that are a functional intermediate of the ER-to-Golgi transport in yeast

The SAR1 gene product (Sar1p), a 21-kD GTPase, is a key component of the ER-to-Golgi transport in the budding yeast. We previously reported that the in vitro reconstitution of protein transport from the ER to the Golgi was dependent on Sar1p and Sec12p (Oka, T., S. Nishikawa, and A. Nakano. 1991. J. Cell Biol. 114:671-679). Sec12p is an integral membrane protein in the ER and is essential for the Sar1 function. In this paper, we show that Sar1p can remedy the temperature-sensitive defect of the sec12 mutant membranes, which is in the formation of ER- to-Golgi transport vesicles. The addition of Sar1p promotes vesicle formation from the ER irrespective of the GTP- or GTP gamma S-bound form, indicating that the active form of Sar1p but not the hydrolysis of GTP is required for this process. The inhibition of GTP hydrolysis blocks transport of vesicles to the Golgi and thus causes their accumulation. The accumulating vesicles, which carry Sar1p on them, can be separated from other membranes, and, after an appropriate wash that removes Sar1p, are capable of delivering the content to the Golgi when added back to fresh membranes. Thus we have established a new method for isolation of functional intermediate vesicles in the ER-to-Golgi transport. The sec23 mutant is defective in activation of Sar1 GTPase (Yoshihisa, T., C. Barlowe, and R. Schekman. 1993. Science (Wash. DC). 259:1466-1468). The membranes and cytosol from the sec23 mutant show only a partial defect in vesicle formation and this defect is also suppressed by the increase of Sar1p. Again GTP hydrolysis is not needed for the suppression of the defect in vesicle formation. Based on these results, we propose a model in which Sar1p in the GTP-bound form is required for the formation of transport vesicles from the ER and the GTP hydrolysis by Sar1p is essential for entering the next step of vesicular transport to the Golgi apparatus.     

In vitro reconstitution of intercompartmental protein transport to the yeast vacuole

Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.     

Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights approximately equal to 20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H]mannose and their sensitivity to endoglycosidase H. The fourth protein (molecular weight approximately equal to 30,000) also was labeled with [3H]mannose but was insensitive to endoglycosidase H; it appeared to contain O-linked sugars. In sec mutants accumulating Golgi membranes or post-Golgi vesicles, a 35-kilodalton protein was labeled with [3H]palmitate. Analysis of Staphylococcus aureus protease V8 digests and pulse-chase experiments indicated that the 30-kilodalton protein was a precursor of 35 kilodaltons. None of these proteins was labeled with [3H]palmitate in a sec mutant that blocked the penetration of nascent polypeptides into endoplasmic reticulum; thus, acylation occurred in endoplasmic reticulum. All four proteins could be recovered from fractions enriched for yeast membranes. Fatty acids were not released from proteins by boiling in sodium dodecyl sulfate or extraction with organic solvents but were recovered as methyl esters after proteins were treated with KOH-methanol, a reaction characteristic of an acyl ester linkage.     

Sec24p and Iss1p function interchangeably in transport vesicle formation from the endoplasmic reticulum in Saccharomyces cerevisiae

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations in SEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.