Yeast Snc proteins complex with Sec9. Functional interactions between putative SNARE proteins

Yeast possess two homologs of the synaptobrevin family of vesicle-associated proteins that are proposed to be involved in membrane recognition and to act as receptors for components of the fusion machinery in neurons. We have previously described the yeast homologs, Snc1 and Snc2, and demonstrated that they localize to secretory vesicles and are required for normal secretion. Yeast lacking Snc protein expression accumulate post-Golgi transport vesicles that contain secretory proteins. Therefore, Snc proteins are essential for the fusion of carrier vesicles with the plasma membrane, and this property appears to have been conserved in evolution. We have now examined whether Snc proteins interact with other components of the late secretory pathway in yeast. Here we show that Snc proteins form a tight genetic and physical interaction with a plasma membrane protein, Sec9. Sec9 is the yeast equivalent of SNAP-25, a second receptor protein from neurons that has been shown to interact with synaptobrevin. We suggest, then, that recognition of the plasma membrane by secretory vesicles may involve the formation of a Snc-Sec9 complex and that this interaction has evolved as a fundamental step in secretory processes.     

Involvement of long chain fatty acid elongation in the trafficking of secretory vesicles in yeast

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.     

Hebb and Darwin

Like in animal cells, the major secretory pathway of the ascomycetous budding yeast Saccharomyces (s.) cerevisiae consists of membrane-bound compartments which transport soluble and membrane (glyco)peptides to lysosomal vacuoles, cell wall, or out of the cell. The established model of the cellular machinery of the yeast secretory pathway was deduced largerly from molecular ex situ analyses and for budding yeast cells it was assumed to be identical with that of secretory animal cells. Interphase yeast cells were never considered. Glycosylation of peptides was detected in the endoplasmic reticulum (ER) and the putative Golgi cisternae. Coated membrane vesicles were assumed to transport intermediates into and within the Golgi cascade. Proteolytic trimming would occur in the last Golgi compartment. Golgi-derived membrane vesicles would serve for exocytosis or fuse with lysosomal vacuoles. In contrast to this notion, yeast cytologists showed specific features of secretion in S. cerevisiae and other Ascomycetes. Cytochemical observations in situ of both dividing and interphase yeast showed direct communication between nuclear envelope, ER and segregated Golgi cisternae. A new class of constitutive conveyors, coated protein globules smaller than membrane vesicles, was shown to exist throughout the cell cycle. The function of Golgi-derived membrane vesicles was constrained to promotion of exocytosis in budding yeast. Some of the Golgi apparatus functions were detected in both these classes of exocytotic conveyors. Uptake (phagocytosis) of transport conveyors and lipoprotein condensates has been shown to deliver enzymes and secretory compounds into vacuoles. This simplified machinery of secretion, postulated for S. cerevisiae, does not include the Golgi cascade.     

The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.     

Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway

The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.     

Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Delta strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Delta yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

A link between secretion and pre-mRNA processing defects in Saccharomyces cerevisiae and the identification of a novel splicing gene, RSE1

Secretory proteins in eukaryotic cells are transported to the cell surface via the endoplasmic reticulum (ER) and the Golgi apparatus by membrane-bounded vesicles. We screened a collection of temperature-sensitive mutants of Saccharomyces cerevisiae for defects in ER-to-Golgi transport. Two of the genes identified in this screen were PRP2, which encodes a known pre-mRNA splicing factor, and RSE1, a novel gene that we show to be important for pre-mRNA splicing. Both prp2-13 and rse1-1 mutants accumulate the ER forms of invertase and the vacuolar protease CPY at restrictive temperature. The secretion defect in each mutant can be suppressed by increasing the amount of SAR1, which encodes a small GTPase essential for COPII vesicle formation from the ER, or by deleting the intron from the SAR1 gene. These data indicate that a failure to splice SAR1 pre-mRNA is the specific cause of the secretion defects in prp2-13 and rse1-1. Moreover, these data imply that Sar1p is a limiting component of the ER-to-Golgi transport machinery and suggest a way that secretory pathway function might be coordinated with the amount of gene expression in a cell.     

Vesicle budding on Golgi membranes: regulation by G proteins and myosin motors

One of the main functions of the Golgi complex is to generate transport vesicles for the post-Golgi trafficking of proteins in secretory pathways. Many different populations of vesicles are distinguished by unique sets of structural and regulatory proteins which participate in vesicle budding and fusion. Monomeric and heterotrimeric G proteins regulate vesicle budding and secretory traffic into and out of the Golgi complex. An inventory of G protein alpha subunits associated with Golgi membranes highlights their diverse involvement and potential for coupling Golgi trafficking, through various signal transduction pathways, to cell growth or other more specialized cell functions. Cytoskeletal proteins are now also known to associate specifically with the Golgi complex and Golgi-derived vesicles. Amongst these, conventional and unconventional myosins are recruited to vesicle membranes. Several roles in vesicle budding and vesicle trafficking can be proposed for these actin-based motors.     

Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.     

Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex

In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod approximately 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.     

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in alpha-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.     

ATP-dependent transport of reduced glutathione on YCF1, the yeast orthologue of mammalian multidrug resistance associated proteins

The transport systems involved in the export of cellular reduced glutathione (GSH) have not been identified, although recent studies implicate a role for some of the multidrug resistance associated proteins (MRP), including MRP1 and MRP2. The present study examined the hypothesis that the yeast orthologue of MRP, Ycf1p, mediates ATP-dependent GSH transport. [3H]GSH transport was measured in vacuolar membrane vesicles isolated from a control strain of Saccharomyces cerevisiae (DTY165), the isogenic DTY167 strain that lacks a functional Ycf1p, and in DTY167 transformed with a 2-micrometer plasmid vector containing YCF1. GSH transport in control vacuolar membrane vesicles was mediated largely by an ATP-dependent, low affinity pathway (Km = 15 +/- 4 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast Ycf1p transporter and inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, probenecid, and sulfinpyrazone, inhibitors of MRP1 and MRP2, but was minimally affected by membrane potential or pH gradient uncouplers. In contrast, ATP-dependent GSH transport was not seen in vacuolar membrane vesicles isolated from the DTY167 yeast strain without a functional Ycf1p but was restored to near wild-type levels in the DTY167 strain transformed with YCF1 and expressing the vacuolar Ycf1p transporter. On the other hand, expression and functional activity of a bile acid transporter, Bat1p, and of the V-type ATPase were similar in all three yeast strains. These results provide direct evidence for ATP-dependent low affinity transport of GSH by the yeast Ycf1p transporter. Because of the structural and functional homology between Ycf1p and MRP1 and MRP2, these data support the hypothesis that GSH efflux from mammalian cells is mediated by these membrane proteins.     

Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive. With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable. To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed. A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected. Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.     

Biosynthetic protein transport through the early secretory pathway

Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation.     

Aut2p and Aut7p, two novel microtubule-associated proteins are essential for delivery of autophagic vesicles to the vacuole

AUT2 and AUT7, two novel genes essential for autophagocytosis in the yeast Saccharomyces cerevisiae were isolated. AUT7 was identified as a low copy suppressor of autophagic defects in aut2-1 cells. Aut7p is a homologue of the rat microtubule-associated protein (MAP) light chain 3 (LC3). Aut2p and Aut7p interact physically. Aut7p is attached to microtubules via Aut2p, which interacts with tubulins Tub1p and Tub2p. aut2- and aut7-deleted cells are unable to deliver autophagic vesicles and the precursor of aminopeptidase I to the vacuole. Double membrane-layered autophagosome-like vesicles accumulate in the cytoplasm of these cells. Our findings suggest that microtubules and an attached protein complex of Aut2p and Aut7p are involved in the delivery of autophagic vesicles to the vacuole.     

Characteristics of endoplasmic reticulum-derived transport vesicles

We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules.     

Identification of the MNN2 and MNN5 mannosyltransferases required for forming and extending the mannose branches of the outer chain mannans of Saccharomyces cerevisiae

The mannan structure found on the N-linked glycans of the yeast Saccharomyces cerevisiae is composed of a long backbone of alpha-1, 6-linked mannose to which are attached branches consisting of two alpha-1,2-linked mannoses followed by an alpha-1,3-linked mannose. In the mutants mnn2 and mnn5, the addition of the first and second of these two mannoses, respectively, is defective. In this paper, we report the identification of the genes corresponding to these mutations. The two genes encode closely related proteins with distant homology to the known Mnn1p alpha-1,3-mannosyltransferase. We show that these proteins are localized in an early compartment of the yeast Golgi and that they are not physically associated with each other or with the two protein complexes known to be involved in synthesizing the alpha-1,6-linked backbone. The identification of Mnn2p and Mnn5p allows us to assign Golgi proteins to all of the catalytic steps in S. cerevisiae mannan synthesis.     

Simultaneous determination of cytotoxic (adriamycin, vincristine) and modulator of resistance (verapamil, S 9788) drugs in human cells by high-performance liquid chromatography and ultraviolet detection

We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51-56]. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.     

Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (. Genetics. 142:393-406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE-associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.