The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.     

Concentration of GPI-Anchored Proteins upon ER Exit in Yeast

Previous biochemical work has revealed two parallel routes of exit from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae , one seemingly specific for glycosyl-phosphatidylinositol (GPI)-anchored proteins. Using the coat protein II (COPII) mutant sec31-1 , we visualized ER exit sites (ERES) and identified three distinct ERES populations in vivo. One contains glycosylated pro-α-factor, the second contains the GPI-anchored proteins Cwp2p, Ccw14p and Tos6p and the third is enriched with the hexose transporter, Hxt1p. Concentration of GPI-anchored proteins prior to budding requires anchor remodeling, and Hxt1p incorporation into ERES requires the COPII components Sec12p and Sec16p. Additionally, we have found that GPI-anchored protein ER exit is controlled by the p24 family member Emp24p, whereas ER export of most transmembrane proteins requires the Cornichon homologue Erv14p.     

Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a posttranslational modification occurring in the endoplasmic reticulum (ER). After GPI attachment, proteins are transported by coat protein complex II (COPII)-coated vesicles from the ER. Because GPI-anchored proteins (GPI-APs) are localized in the lumen, they cannot interact with cytosolic COPII components directly. Receptors that link GPI-APs to COPII are thought to be involved in efficient packaging of GPI-APs into vesicles; however, mechanisms of GPI-AP sorting are not well understood. Here we describe two remodeling reactions for GPI anchors, mediated by PGAP1 and PGAP5, which were required for sorting of GPI-APs to ER exit sites. The p24 family of proteins recognized the remodeled GPI-APs and sorted them into COPII vesicles. Association of p24 proteins with GPI-APs was pH dependent, which suggests that they bind in the ER and dissociate in post-ER acidic compartments. Our results indicate that p24 complexes act as cargo receptors for correctly remodeled GPI-APs to be sorted into COPII vesicles.     

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.     

The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles

Members of the yeast p24 family, including Emp24p and Erv25p, form a heteromeric complex required for the efficient transport of selected proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The specific functions and sites of action of this complex are unknown. We show that Emp24p is directly required for efficient packaging of a lumenal cargo protein, Gas1p, into ER-derived vesicles. Emp24p and Erv25p can be directly cross-linked to Gas1p in ER-derived vesicles. Gap1p, which was not affected by emp24 mutation, was not cross-linked. These results suggest that the Emp24 complex acts as a cargo receptor in vesicle biogenesis from the ER.     

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.     

The Rab GTPase Ypt1p and tethering factors couple protein sorting at the ER to vesicle targeting to the Golgi apparatus

GPI-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event can be reproduced in vitro. When extracts from a uso1 mutant were used, the sorting of GPI-anchored proteins from other secretory proteins was defective. Complementation with purified Uso1p restored sorting. The Rab GTPase Ypt1p and the tethering factors Sec34p and Sec35p, but not Bet3p, a member of the TRAPP complex, were also required for protein sorting upon ER exit. Therefore, the Ypt1p tethering complex couples protein sorting in the ER to vesicle targeting to the Golgi apparatus. Sorting of GPI-anchored proteins from other secretory proteins was also observed in vivo. The sorting defect observed in vitro with uso1 and ypt1 mutants was reproduced in vivo.     

Erv26p-Dependent Export of Alkaline Phosphatase from the ER Requires Lumenal Domain Recognition

Active sorting at the endoplasmic reticulum (ER) drives efficient export of fully folded secretory proteins into coat protein complex II (COPII) vesicles, whereas ER-resident and misfolded proteins are retained and/or degraded. A number of secretory proteins depend upon polytopic cargo receptors for linkage to the COPII coat and ER export. However, the mechanism by which cargo receptors recognize transport-competent cargo is poorly understood. Here we examine the sorting determinants required for export of yeast alkaline phosphatase (ALP) by its cargo receptor Erv26p. Analyses of ALP chimeras and mutants indicated that Erv26p recognizes sorting information in the lumenal domain of ALP. This lumenal domain sorting signal must be positioned near the inner leaflet of the ER membrane for Erv26p-dependent export. Moreover, only assembled ALP dimers were efficiently recognized by Erv26p while an ALP mutant blocked in dimer assembly failed to exit the ER and was subjected to ER-associated degradation. These results further refine sorting information for ER export of ALP and show that recognition of folded cargo by export receptors contributes to strict ER quality control.     

Concentrative sorting of secretory cargo proteins into COPII-coated vesicles

Here, we show that efficient transport of membrane and secretory proteins from the ER of Saccharomyces cerevisiae requires concentrative and signal-mediated sorting. Three independent markers of bulk flow transport out of the ER indicate that in the absence of an ER export signal, molecules are inefficiently captured into coat protein complex II (COPII)-coated vesicles. A soluble secretory protein, glycosylated pro-alpha-factor (gpalphaf), was enriched approximately 20 fold in these vesicles relative to bulk flow markers. In the absence of Erv29p, a membrane protein that facilitates gpalphaf transport (Belden and Barlowe, 2001), gpalphaf is packaged into COPII vesicles as inefficiently as soluble bulk flow markers. We also found that a plasma membrane protein, the general amino acid permease (Gap1p), is enriched approximately threefold in COPII vesicles relative to membrane phospholipids. Mutation of a diacidic sequence present in the COOH-terminal cytosolic domain of Gap1p eliminated concentrative sorting of this protein.     

The yeast p24 complex regulates GPI-anchored protein transport and quality control by monitoring anchor remodeling

Glycosylphosphatidylinositol (GPI)-anchored proteins are secretory proteins that are attached to the cell surface of eukaryotic cells by a glycolipid moiety. Once GPI anchoring has occurred in the lumen of the endoplasmic reticulum (ER), the structure of the lipid part on the GPI anchor undergoes a remodeling process prior to ER exit. In this study, we provide evidence suggesting that the yeast p24 complex, through binding specifically to GPI-anchored proteins in an anchor-dependent manner, plays a dual role in their selective trafficking. First, the p24 complex promotes efficient ER exit of remodeled GPI-anchored proteins after concentration by connecting them with the COPII coat and thus facilitates their incorporation into vesicles. Second, it retrieves escaped, unremodeled GPI-anchored proteins from the Golgi to the ER in COPI vesicles. Therefore the p24 complex, by sensing the status of the GPI anchor, regulates GPI-anchored protein intracellular transport and coordinates this with correct anchor remodeling.     

The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER

Erv41p and Erv46p form an integral membrane protein complex that cycles between the endoplasmic reticulum (ER) and Golgi. Both proteins contain a large lumenal domain and short N- and C-terminal tail sequences exposed to the cytosol. The coat protein complex II (COPII) packages the Erv41p-Erv46p complex into ER-derived vesicles for delivery to the Golgi. We determined signals in the Erv41p-Erv46p complex that are required for COPII-dependent export from the ER. Mutants lacking the Erv41p or Erv46p C-terminus accumulated in the ER and were not packaged efficiently into vesicles. We identified an isoleucine-leucine sequence in the Erv41p tail that was required for COPII binding and inclusion of the complex into vesicles. This signal was sufficient for COPII binding but not for ER export. The Erv46p tail contains a phenylalanine-tyrosine sequence required together with the isoleucine-leucine signal in Erv41p for export of the complex. Surprisingly, Erv41p- Erv46p tail-swapped chimeras were not exported from the ER, indicating that signals in both the Erv41p and the Erv46p tail sequences are required in a specific orientation for efficient packaging of the Erv41p-Erv46p complex.     

Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi

Endoplasmic reticulum (ER) quality control (ERQC) components retain and degrade misfolded proteins, and our results have found that the degradation of the soluble ERQC substrates CPY* and PrA* but not membrane spanning ERQC substrates requires transport between the ER and Golgi. Stabilization of these misfolded soluble proteins was seen in cells lacking Erv29p, a probable Golgi localized protein that cycles through the ER by means of a di-lysine ER retrieval motif (KKKIY). Cells lacking Erv29p also displayed severely retarded ER exit kinetics for a subset of correctly folded proteins. We suggest that Erv29p is likely involved in cargo loading of a subset of proteins, including soluble misfolded proteins, into vesicles for ER exit. The stabilization of soluble ERQC substrates in both erv29Delta cells and sec mutants blocked in either ER exit (sec12) or vesicle delivery to the Golgi (sec18) suggests that ER-Golgi transport is required for ERQC and reveals a new aspect of the degradative mechanism.     

Sorting signals can direct receptor-mediated export of soluble proteins into COPII vesicles

Soluble secretory proteins are first translocated across endoplasmic reticulum (ER) membranes and folded in a specialized ER luminal environment. Fully folded and assembled secretory cargo are then segregated from ER-resident proteins into COPII-derived vesicles or tubular elements for anterograde transport. Mechanisms of bulk-flow, ER-retention and receptor-mediated export have been suggested to operate during this transport step, although these mechanisms are poorly understood. In yeast, there is evidence to suggest that Erv29p functions as a transmembrane receptor for the export of certain soluble cargo proteins including glycopro-alpha-factor (gpalphaf), the precursor of alpha-factor mating pheromone. Here we identify a hydrophobic signal within the pro-region of gpalphaf that is necessary for efficient packaging into COPII vesicles and for binding to Erv29p. When fused to Kar2p, an ER-resident protein, the pro-region sorting signal was sufficient to direct Erv29p-dependent export of the fusion protein into COPII vesicles. These findings indicate that specific motifs within soluble secretory proteins function in receptor-mediated export from the ER. Moreover, positive sorting signals seem to predominate over potential ER-retention mechanisms that may operate in localizing ER-resident proteins such as Kar2p.     

Inositol deacylation by Bst1p is required for the quality control of glycosylphosphatidylinositol-anchored proteins

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins. We have constructed a mutant form of the beta-1,3-glucanosyltransferase Gas1p (Gas1*p) as a model misfolded GPI-anchored protein. Gas1*p was modified with a GPI anchor but retained in the ER and was degraded rapidly via the proteasome. Disruption of BST1, which encodes GPI inositol deacylase, caused a delay in the degradation of Gas1*p. This delay was because of an effect on the deacylation activity of Bst1p. Disruption of genes involved in GPI-anchored protein concentration and N-glycan processing caused different effects on the degradation of Gas1*p and a soluble misfolded version of carboxypeptidase Y. Furthermore, Gas1*p associated with both Bst1p and BiP/Kar2p, a molecular chaperone, in vivo. Our data suggest that GPI inositol deacylation plays important roles in the quality control and ER-associated degradation of GPI-anchored proteins.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER). After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profile of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchor-deficient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-deficient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by the transport of GPI anchor molecules.     

Apolipoprotein B100 exit from the endoplasmic reticulum (ER) is COPII-dependent,and its lipidation to very low density lipoprotein occurs post-ER

Hepatic apolipoprotein B100 (apoB100) associates with lipids to form dense lipoprotein particles in the endoplasmic reticulum (ER) and is further lipidated to very low density lipoproteins (VLDL). Because the VLDL diameter can exceed 200 nm, classical ER-derived vesicles may be unable to accommodate VLDLs. Using hepatic membranes and cytosol to reconstitute ER budding, apoB100-containing vesicles sedimented distinct from those harboring more typical cargo but contained Sec23. Moreover, ER exit of apoB was inhibited by dominant-negative Sar1. Budding required Sar1 regardless of whether oleic acid (OA) was added to stimulate apoB lipidation; therefore, either large apoB100-lipoproteins reside in secretory vesicles, or full lipidation occurs post-ER. Using membranes from cells incubated in the presence or absence of OA, we determined that apoB100-lipoproteins in ER vesicles had not become lipidated to VLDLs. VLDL particles resided in the Golgi, but not the ER, after fractionation of OA-treated cells. We conclude that apoB100-lipoproteins exit the ER in COPII vesicles, but under conditions favorable for VLDL formation final lipid loading occurs post-ER.     

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.     

Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities

Efficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport.Anterograde transport in the eukaryotic secretory pathway is initiated by the formation of COPII²-coated vesicles that emerge from transitional ER sites. The COPII coat, which consists of the small GTPase Sar1p, Sec23/24 complex, and Sec13/31 complex, selects vesicle cargo through recognition of export signals and forms ER-derived vesicles through assembly of an outer layer cage structure (1, 2). Cytoplasmically exposed ER export signals have been identified in secretory cargo including the C-terminal dihydrophic and diacidic motifs (3, 4). Structural studies indicate that the Sec24p subunit of the COPII coat contains distinct binding sites for some of the molecularly defined export signals (5, 6). Thus a cycle of cargo-coat interactions regulated by the Sar1p GTPase directs anterograde movement of secretory proteins into ER-derived transport vesicles (7).Although many secretory proteins contain known export signals that interact directly with COPII subunits, the diverse array of secretory cargo that depends on this export route requires additional machinery for efficient collection of all cargo into COPII vesicles (1). For instance certain soluble secretory proteins as well as transmembrane cargo require protein sorting adaptors for efficient ER export. These membrane-spanning adaptors, or sorting receptors, interact directly with secretory cargo and with coat subunits to efficiently couple cargo to the COPII budding machinery. For example, ERGIC-53 acts as a protein sorting adaptor for several glycoproteins and has a large N-terminal lumenal domain that interacts with secretory proteins including blood coagulation factors, cathepsins, and α₁-antitrypsin (8–10). The cytoplasmic C-terminal tail of ERGIC-53 contains a diphenylalanine export signal that is necessary for COPII export as well as a dilysine motif required for COPI-dependent retrieval to the ER (11). Additional ER vesicle proteins identified in yeast have been shown to interact with the COPII coat as well as specific secretory proteins (12). For example Erv29p acts as a protein sorting adaptor for the soluble secretory proteins glyco-pro-α-factor and carboxypeptidase Y (13). Erv29p also contains COPII and COPI sorting signals that shuttle the protein between ER and Golgi compartments. More recently Erv26p was identified as a cargo receptor that escorts the pro-form of secretory alkaline phosphatase (ALP) into COPII-coated vesicles (14).Although COPII sorting receptors have been identified, the molecular mechanisms by which these receptors link cargo to coat remain poorly understood. Moreover it is not clear how cargo binding is regulated to promote interaction in the ER and then trigger dissociation in the Golgi complex. We have shown previously that Erv26p binds to pro-ALP and is required for efficient export of this secretory protein from the ER (14). Therefore specific lumenal regions of Erv26p are proposed to interact with pro-ALP, whereas cytosolically exposed sorting signals are presumably recognized and bound by coat subunits. To gain insight on the molecular contacts required for Erv26p sorting function, we undertook a systematic mutational analysis of this multispanning membrane protein. After generating a series of Erv26p mutants, we observed that mutation of specific residues in the third loop domain affect pro-ALP interaction and that residues in the C-terminal cytosolic tail are required for COPII and COPI transport. Finally mutation of residues in the second loop domain influenced Erv26p homodimer formation and sorting activity.     

ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains

Protein transport between the ER and the Golgi in mammalian cells occurs via large pleiomorphic carriers, and most current models suggest that these are formed by the fusion of small ER-derived COPII vesicles. We have examined the dynamics and structural features of these carriers during and after their formation from the ER by correlative video/light electron microscopy and tomography. We found that saccular carriers containing either the large supramolecular cargo procollagen or the small diffusible cargo protein VSVG arise through cargo concentration and direct en bloc protrusion of specialized ER domains in the vicinity of COPII-coated exit sites. This formation process is COPII dependent but does not involve budding and fusion of COPII-dependent vesicles. Fully protruded saccules then move centripetally, evolving into one of two types of carriers (with distinct kinetic and structural features). These findings provide an alternative framework for analysis of ER-to-Golgi traffic.