Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery

The roles of the components of the Sec34p protein complex in intracellular membrane trafficking, first identified in the yeast Saccharomyces cerevisiae, have yet to be characterized in higher eukaryotes. We cloned a human cDNA whose predicted amino acid sequence showed 41% similarity to yeast Sec34p with homology throughout the entire coding region. Affinity-purified antibodies raised against the human SEC34 protein (hSec34p) recognized a cellular protein of 94 kDa in both soluble and membrane fractions. Like yeast Sec34p, cytosolic hSec34p migrated with an apparent molecular mass of 300 kDa on a glycerol velocity gradient, suggesting that it is part of a protein complex. Immunofluorescence microscopy localized hSec34p to the Golgi compartment in cells of all species examined, where it co-localized well with the cis/medial Golgi marker membrin and partially co-localized with cis-Golgi network marker p115 and trans-Golgi marker TGN38. The co-localization with membrin was maintained at 15 degrees C and after microtubule depolymerization with nocodazole. During transport of the tsO45 vesicular stomatitis virus G protein through the Golgi, there was significant overlap with the hSec34p compartment. Green fluorescent protein-hSec34 expressed in HeLa cells was restricted to Golgi cisternae, and its membrane association was sensitive to brefeldin A treatment. Taken together, our findings indicate that hSec34p is part of a peripheral membrane protein complex localized on cis/medial Golgi cisternae where it may participate in tethering intra-Golgi transport vesicles.     

Identification of Sec36p,Sec37p,and Sec38p: components of yeast complex that contains Sec34p and Sec35p

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.     

Sgf1p, a New Component of the Sec34p/Sec35p Complex

Here we report the identification of SGF1 as a high-copy suppressor of the sec35–1 mutant. SGF1 encodes an essential hydrophilic protein of ∼ 100 kDa. Using the yeast two-hybrid system and coprecipitation studies, we demonstrate that Sgf1p is a new subunit of the multiprotein Sec34p/Sec35p complex. Reduced levels of Sgf1p lead to the accumulation of a variety of membranes as well as a kinetic block in endoplasmic reticulum to Golgi traffic. Immunofluorescence studies demonstrate that Sec34p is found throughout the Golgi, with a high concentration on early Golgi. Although an earlier study suggested that Sec34p (Grd20p) is not required for protein secretion, we show here that the sec34–2 and sec35–1 mutations lead to a pleiotropic block in the secretion of all proteins into the growth medium.     

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.     

High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Delta mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of approximately 100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of approximately 480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.     

The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic.

The specificity of intracellular vesicle transport is mediated in part by tethering factors that attach the vesicle to the destination organelle prior to fusion. We have identified a protein, Dor1p, that is involved in vesicle targeting to the yeast Golgi apparatus and found it to be associated with seven further proteins. Identification of these revealed that they include Sec34p and Sec35p, the two known components of the Sec34/35 complex previously proposed to tether vesicles to the Golgi. Of the six previously uncharacterized components, four have homologs in higher eukaryotes, including a subunit of a mammalian Golgi transport complex. Furthermore, several of the proteins show distant homology to components of two other putative tethering complexes, the exocyst and the Vps52/53/54 complex, revealing that tethering factors involved in different membrane traffic steps are structurally related.     

Arabidopsis Sec21p and Sec23p homologs. Probable coat proteins of plant COP-coated vesicles

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.     

Sec22p export from the endoplasmic reticulum is independent of SNARE pairing

Molecularly distinct sets of SNARE proteins localize to specific intracellular compartments and catalyze membrane fusion events. Although their central role in membrane fusion is appreciated, little is known about the mechanisms by which individual SNARE proteins are targeted to specific organelles. Here we investigated functional domains in Sec22p that direct this SNARE protein to the endoplasmic reticulum (ER), to Golgi membranes, and into SNARE complexes with Bet1p, Bos1p, and Sed5p. A series of Sec22p deletion mutants were monitored in COPII budding assays, subcellular fractionation gradients, and SNARE complex immunoprecipitations. We found that the N-terminal "profilin-like" domain of Sec22p was required but not sufficient for COPII-dependent export of Sec22p from the ER. Interestingly, versions of Sec22p that lacked the N-terminal domain were assembled into ER/Golgi SNARE complexes. Analyses of Sec22p SNARE domain mutants revealed a second signal within the SNARE motif (between layers -4 and -1) that was required for efficient ER export. Other SNARE domain mutants that contained this signal were efficiently packaged into COPII vesicles but failed to assemble into SNARE complexes. Together these results indicated that SNARE complex formation is neither required nor sufficient for Sec22p packaging into COPII transport vesicles and subsequent targeting to the Golgi complex. We propose that the COPII budding machinery has a preference for unassembled ER/Golgi SNARE proteins.     

Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp

Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus.     

Sec14 homology domain targets p50RhoGAP to endosomes and provides a link between Rab and Rho GTPases

Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.     

p125A exists as part of the mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport

Coat protein II (COPII)–mediated export from the endoplasmic reticulum (ER) involves sequential recruitment of COPII complex components, including the Sar1 GTPase, the Sec23/Sec24 subcomplex, and the Sec13/Sec31 subcomplex. p125A was originally identified as a Sec23A-interacting protein. Here we demonstrate that p125A also interacts with the C-terminal region of Sec31A. The Sec31A-interacting domain of p125A is between residues 260–600, and is therefore a distinct domain from that required for interaction with Sec23A. Gel filtration and immunodepletion studies suggest that the majority of cytosolic p125A exists as a ternary complex with the Sec13/Sec31A subcomplex, suggesting that Sec 13, Sec31A, and p125A exist in the cytosol primarily as preassembled Sec13/Sec31A/p125A heterohexamers. Golgi morphology and protein export from the ER were affected in p125A-silenced cells. Our results suggest that p125A is part of the Sec13/Sec31A subcomplex and facilitates ER export in mammalian cells.     

Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. "Deep etch" EM of purified COG revealed an approximately 37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.     

p125 is a novel mammalian Sec23p-interacting protein with structural similarity to phospholipid-modifying proteins.

COPII-coated vesicles are involved in protein transport from the endoplasmic reticulum to the Golgi apparatus. COPII consists of three parts: Sar1p and the two protein complexes, Sec23p-Sec24p and Sec13p-Sec31p. Using a glutathione S-transferase fusion protein with mouse Sec23p, we identified a novel mammalian Sec23p-interacting protein, p125, which is clearly distinct from Sec24p. The N-terminal region of p125 is rich in proline residues, and the central and C-terminal regions exhibit significant homology to phospholipid-modifying proteins, especially phosphatidic acid preferring-phospholipase A1. We transiently expressed p125 and mouse Sec23p in mammalian cells and examined their interaction. The results showed that the N-terminal region of p125 is important for the interaction with Sec23p. We confirmed the interaction between the two proteins by a yeast two-hybrid assay. Overexpression of p125, like that of mammalian Sec23p, caused disorganization of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus, suggesting its role in the early secretory pathway.     

Evidence for overlapping and distinct functions in protein transport of coat protein Sec24p family members

Budding of transport vesicles from the endoplasmic reticulum in yeast requires the formation, at the budding site, of a coat protein complex (COPII) that consists of two heterodimeric subcomplexes (Sec23p/Sec24p and Sec13p/Sec31p) and the Sar1 GTPase. Sec24p is an essential protein and involved in cargo selection. In addition to Sec24p, the yeast Saccharomyces cerevisiae expresses two non-essential Sec24p-related proteins, termed Sfb2p (product of YNL049c) and Sfb3p/Lst1p (product of YHR098c). We here show that Sfb2p and, less efficiently, Sfb3p/Lst1p are able to bind, like Sec24p, the integral membrane cargo protein Sed5p. We also demonstrate that Sfb2p, like Sec24p and Sfb3p/Lst1p, forms a complex with Sec23p in vivo. Whereas the deletion of SFB2 did not affect transport kinetics of various proteins, the maturation of the glycolipid-anchored plasma membrane protein Gas1p was differentially impaired in sfb3 knock-out cells. We generated several conditional-lethal sec24 mutants that, combined with null alleles of SFB2 and SFB3/LST1, led to a complete block of transport between the endoplasmic reticulum and the Golgi (sec24-11/Deltasfb2) or to cell death (sec24-11/Deltasfb3). Of the Sec24p family members, Sfb2p is the least abundant at steady state, but high intracellular concentrations of Sfb2p can rescue sec24 mutants under restrictive conditions. The data presented strongly suggest that the Sec24p-related proteins function as COPII components.     

Mammalian Sec61 is associated with Sec62 and Sec63

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.     

COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.

Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.     

Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms: Rer1p-dependent retrieval that requires the transmembrane domain and Rer1p-independent retention that involves the cytoplasmic domain

Yeast Sec12p is a type II transmembrane protein in the ER, which is essential for the formation of transport vesicles. From biochemical and morphological lines of evidence, we have proposed that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early Golgi compartment. We have also shown that Rer1p, a membrane protein in the Golgi, is required for correct localization of Sec12p. In the present study, we have performed a systematic analysis to determine the ER localization signals in Sec12p corresponding to these two mechanisms. Both the transmembrane domain (TMD) and the NH2-terminal cytoplasmic domain of Sec12p show the ability to localize the protein to the ER. The effect of the TMD is potent and sufficient by itself for the ER localization and is strongly dependent on Rer1p. On the other hand, the cytoplasmic domain shows a moderate ER-localization capability which is independent of Rer1p. The rate of mannosyl modification has been measured to distinguish between retention and retrieval. The cytoplasmic domain significantly delays the transport from the ER to the cis-Golgi. In contrast, the TMD shows only a subtle retardation in the transport from the ER to the cis-Golgi but strictly prevents the transport beyond there. From these observations, we conclude that the TMD mainly acts as the retrieval signal and the cytoplasmic domain contains the retention signal. This study not only supports the two-mechanisms hypothesis but also provides powerful tools to dissect the two.     

A novel phospholipase A1 with sequence homology to a mammalian Sec23p-interacting protein, p125.

p125, a mammalian Sec23p-interacting protein, exhibits sequence homology with bovine testis phosphatidic acid-preferring phospholipase A(1). In this study, we identified and characterized a new homologue of p125, KIAA0725p. KIAA0725p exhibited remarkable sequence similarity with p125 throughout the entire sequence determined but lacked an N-terminal proline-rich, Sec23p-interacting region. In vitro binding analysis showed that KIAA0725p does not bind to Sec23p. KIAA0725p possessed phospholipase A(1) activity preferentially for phosphatidic acid. We examined the effects of overexpression of KIAA0725p on the morphology of organelles. Overexpression of KIAA0725p, like that of p125, caused dispersion of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins located in the Golgi region and caused aggregation of the endoplasmic reticulum. Our results indicate that KIAA0725p is a new member of the phosphatidic acid-preferring phospholipase A(1) protein family and suggest that the cellular function of KIAA0725p is different from that of p125.     

Structure-based functional analysis reveals a role for the SM protein Sly1p in retrograde transport to the endoplasmic reticulum

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum.     

The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.

The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport. Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20. This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene. Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex. Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles. We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.