Role of Erv29p in collecting soluble secretory proteins into ER-derived transport vesicles.

Proteins are transported from the endoplasmic reticulum (ER) in vesicles formed by coat protein complex II (COPII). Soluble secretory proteins are thought to leave the ER in these vesicles by "bulk flow" or through recognition by hypothetical shuttling receptors. We found that Erv29p, a conserved transmembrane protein, was directly required for packaging glycosylated pro-alpha-factor (gpalphaf) into COPII vesicles in Saccharomyces cerevisiae. Further, an Erv29p-gpalphaf complex was isolated from ER-derived transport vesicles. In vivo, export of gpalphaf from the ER was saturable and depended on the expression level of Erv29p. These results indicate that membrane receptors can link soluble cargo proteins to the COPII coat.

Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding.

Proteins destined for the secretory pathway must first fold and assemble in the lumen of endoplasmic reticulum (ER). The pathway maintains a quality control mechanism to assure that aberrantly processed proteins are not delivered to their sites of function. As part of this mechanism, misfolded proteins are returned to the cytosol via the ER protein translocation pore where they are ubiquitinated and degraded by the 26S proteasome. Previously, little was known regarding the recognition and targeting of proteins before degradation. By tracking the fate of several mutant proteins subject to quality control, we demonstrate the existence of two distinct sorting mechanisms. In the ER, substrates are either sorted for retention in the ER or are transported to the Golgi apparatus via COPII-coated vesicles. Proteins transported to the Golgi are retrieved to the ER via the retrograde transport system. Ultimately, both retained and retrieved proteins converge at a common machinery at the ER for degradation. Furthermore, we report the identification of a gene playing a novel role specific to the retrieval pathway. The gene, BST1, is required for the transport of misfolded proteins to the Golgi, although dispensable for the transport of many normal cargo proteins.

Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex.

Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K(d) approximately 100 microm), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER.

Deletion of yeast p24 genes activates the unfolded protein response.

Yeast cells lacking a functional p24 complex accumulate a subset of secretory proteins in the endoplasmic reticulum (ER) and increase the extracellular secretion of HDEL-containing ER residents such as Kar2p/BiP. We report that a loss of p24 function causes activation of the unfolded protein response (UPR) and leads to increased KAR2 expression. The HDEL receptor (Erd2p) is functional and traffics in p24 deletion strains as in wild-type strains, however the capacity of the retrieval pathway is exceeded. Other conditions that activate the UPR and elevate KAR2 expression also lead to extracellular secretion of Kar2p. Using an in vitro assay that reconstitutes budding from the ER, we detect elevated levels of Kar2p in ER-derived vesicles from p24 deletion strains and from wild-type strains with an activated UPR. Silencing the UPR by IRE1 deletion diminished Kar2p secretion under these conditions. We suggest that activation of the UPR plays a major role in extracellular secretion of Kar2p.

Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex.

Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain. When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.

Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes.

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.

Organization of the yeast Golgi complex into at least four functionally distinct compartments.

Pro-alpha-factor (pro-alphaf) is posttranslationally modified in the yeast Golgi complex by the addition of alpha1,6-, alpha1,2-, and alpha1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the alpha1,6- and alpha1,3-mannosylation and Kex2p-dependent processing of pro-alphaf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that alpha1,2-mannosylation of pro-alphaf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-alpha1,6-D-mannanase (endoM) were used to quantitate the amount of each pro-alphaf intermediate during transport through the Golgi complex. We found that alpha1,6-, alpha1,2-, and alpha1,3-mannose were sequentially added to pro-alphaf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-alphaf that accumulated in brefeldin A-treated cells received only alpha1,6-mannosylation as did approximately 50% of pro-alphaf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an alpha1,6-mannosyltransferase but lacks alpha1,2-mannosyltransferase activity in vivo. We propose that the alpha1,6-, alpha1,2-, and alpha1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial, trans, and trans-Golgi network of the yeast Golgi complex, respectively.

Traffic COPs of the early secretory pathway.

Intracellular transport between the endoplasmic reticulum and Golgi compartments is mediated by coat protein complexes (COPI and COPII) that form transport vesicles and collect the desired set of cargo. Although the COPI and COPII coats are molecularly distinct, a number of mechanistic parallels appear to be emerging, most notably a general role for small guanine triphosphatases in co-ordinating coat assembly with cargo selection. A combination of morphological, biochemical, and genetic methods is revealing a very dynamic relationship between these compartments, and highlights a central role for COPs in directing traffic through the early secretory pathway. This review focuses on recent advances in molecular mechanisms underlying coated-vesicle assembly and connections with cellular structures.

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.

Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.

Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi.

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.

COPII and selective export from the endoplasmic reticulum.

Forward transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex depends on COPII, a membrane coat that forms ER-derived vesicles. Based on experimental observations, a series of integrated events must be accomplished during the formation of COPII coated vesicles. First, the subunits of the COPII coat must be recruited to the correct site on the surface of the ER. Second, soluble and integral membrane cargo proteins destined for the Golgi complex are concentrated into nascent buds. Third, a set of molecules that must cycle between the ER and Golgi compartments (such as SNARE proteins) are incorporated into vesicles. And fourth, the COPII coat is disassembled after release of ER-derived vesicles thus allowing vesicle fusion and recycling of COPII components. Incorporation of soluble cargo infers the existence of membrane spanning receptor molecules that link lumenal cargo to the vesicle coat. Some candidate proteins have been identified (including the p24 family) that appear to participate in the selection of soluble cargo; however, the mechanistic details of this selection procedure remain obscure. This review will focus on the molecular constituents of the COPII coat and emerging interactions of the coat subunits with proteins involved in selective export from the ER.

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.

Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.

ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy. COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p. LMA1 and Sec18p are required for vesicle fusion after Uso1p function. Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p. Once docked, however, vesicle fusion is no longer sensitive to GDI. In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding to membranes. To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed. These mutations do not interfere with Uso1p-mediated docking, but block membrane fusion. We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.

Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-alpha-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-alpha-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.

Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport.

COPII-coated endoplasmic reticulum (ER)-derived transport vesicles contain a distinct set of membrane-bound polypeptides. We have obtained the NH2-terminal amino acid sequence of polypeptide constituents found on purified vesicles and in this report investigate the 24- and 25-kDa species. The 24-kDa protein is identical to Emp24p, a type I transmembrane protein that is required for transport of a subset of secretory proteins from the ER to the Golgi complex (Schimmöller, F., Singer-Krüger, B., Schröder, S., Krüger, U., Barlowe, C., and Riezman, H. (1995) EMBO J. 14, 1329-1339). The 25-kDa protein, termed Erv25p (ER vesicle protein of 25 kDa), corresponds to an open reading frame found on chromosome XIII of Saccharomyces cerevisiae. Erv25p shares overall sequence identity with Emp24p, but the two proteins are not functionally interchangeable. Antibodies directed against Erv25p reveal that Emp24p and Erv25p depend on each other for stability and form a protein complex that can be isolated after chemical cross-linking. Yeast strains lacking Erv25p (erv25Delta) are viable and display the same selective defect in transport of secretory proteins from the ER to Golgi complex as an emp24Delta strain. A cell-free assay that measures vesicle formation from ER membranes demonstrates that Erv25p and Emp24p are incorporated equally into ER-derived vesicles when COPII-coated budding is reconstituted. Vesicle formation from an erv25Delta strain, an emp24Delta strain and a double erv25Delta emp24Delta strain proceed at wild-type levels; however, incorporation of the Erv25p or the Emp24p protein into COPII-coated vesicles requires expression of both subunits. A potential model for transport of the Erv25p-Emp24p complex between the ER and Golgi compartments is discussed.

Uncoupled packaging of targeting and cargo molecules during transport vesicle budding from the endoplasmic reticulum.

Formation of vesicular intermediates in protein transport between the endoplasmic reticulum and the Golgi apparatus involves a mechanism that sorts and packages two classes of molecules into transport vesicles: targeting molecules, which are required for targeting and consumption of vesicular intermediates, and cargo proteins. In order to examine the importance of cargo in this packaging reaction, we developed an in vitro assay that quantifies vesicle formation based on segregation of targeting molecules. Here we document that endoplasmic reticulum devoid of cargo proteins is competent in the formation and release of targeting molecule-containing vesicles in a fashion indistinguishable from its normal counterpart. This observation implies that packaging of cargo proteins may be uncoupled from the recruitment of targeting molecules during vesicle budding from the endoplasmic reticulum. Using the same assay, we demonstrate that the packaging of targeting molecules into vesicles is not dependent on the lumenal chaperone, BiP (Kar2p).

COPII: a membrane coat that forms endoplasmic reticulum-derived vesicles.

Vesicle budding from the endoplasmic reticulum (ER) has been reconstituted with washed membranes and three soluble proteins: Sec13 complex, Sec23 complex and the small GTPase Sar1p. The proteins that drive this cell-free vesicle budding reaction form an approximately 10 nm thick electron dense coat on ER-derived vesicles. Although the overall mechanism of membrane budding driven by various cytoplasmic coats appears similar, the constituents of this new membrane coat are molecularly distinct from the non-clathrin coat (COP) involved in intra-Golgi transport and the clathrin-containing coats. The new vesicle coat has been termed COPII.

The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi.

Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex. A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl-inositol-anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha-factor, acid phosphatase and two vacuolar proteins is unaffected. Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization. Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII-coated, ER-derived transport vesicles that are isolated from a reconstituted in vitro budding reaction. We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER-derived transport vesicles.