Involvement of the penta-EF-hand protein Pef1p in the Ca2+-dependent regulation of COPII subunit assembly in Saccharomyces cerevisiae

Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca(2+)-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca(2+)-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca(2+) dependence for Sec13/31p association; only the Ca(2+)-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca(2+)-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.     

The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus.

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeast Saccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and higher-molecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ER-Golgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15 degrees C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His6-tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of beta-COP, a component of the COPI complex.     

Human SEC13Rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum

In the yeast Saccharomyces cerevisiae, Sec13p is required for intracellular protein transport from the ER to the Golgi apparatus, and has also been identified as a component of the COPII vesicle coat structure. Recently, a human cDNA encoding a protein 53% identical to yeast Sec13p has been isolated. In this report, we apply the genetic assays of complementation and synthetic lethality to demonstrate the conservation of function between this human protein, designated SEC13Rp, and yeast Sec13p. We show that two reciprocal human/yeast fusion constructs, encoding the NH2-terminal half of one protein and the COOH-terminal half of the other, can each complement the secretion defect of a sec13-1 mutant at 36 degrees C. The chimera encoding the NH2-terminal half of the yeast protein and the COOH-terminal half of the human protein is also able to complement a SEC13 deletion. Overexpression of either the entire human SEC13Rp protein or the chimera encoding the NH2-terminal half of the human protein and the COOH-terminal half of the yeast protein inhibits the growth of a sec13- 1 mutant at 24 degrees C; this growth inhibition is not seen in a wild- type strain nor in other sec mutants, suggesting that the NH2-terminal half of SEC13Rp may compete with Sec13-1p for a common target. We show by immunoelectronmicroscopy of mammalian cells that SEC13Rp (like the putative mammalian homologues of the COPII subunits Sar1p and Sec23p) resides in the region of the transitional ER. We also show that the distribution of SEC13Rp is not affected by brefeldin A treatment. This report presents the first demonstration of a putative mammalian COPII component functioning in yeast, and highlights a potentially useful approach for the study of conserved mammalian proteins in a genetically tractable system.     

Mammalian Sec16/p250 plays a role in membrane traffic from the endoplasmic reticulum

Coat protein complex II (COPII)-coated vesicles/carriers, which mediate export of proteins from the endoplasmic reticulum (ER), are formed at special ER subdomains in mammals, termed ER exit sites or transitional ER. The COPII coat consists of a small GTPase, Sar1, and two protein complexes, Sec23-Sec24 and Sec13-Sec31. Sec23-Sec24 and Sec13-Sec31 appear to constitute the inner and the outermost layers of the COPII coat, respectively. We previously isolated two mammalian proteins (p125 and p250) that bind to Sec23. p125 was found to be a mammalian-specific, phospholipase A(1)-like protein that participates in the organization of ER exit sites. Here we show that p250 is encoded by the KIAA0310 clone and has sequence similarity to yeast Sec16 protein. Although KIAA0310p was found to be localized at ER exit sites, subcellular fractionation revealed its predominant presence in the cytosol. Cytosolic KIAA0310p was recruited to ER membranes in a manner dependent on Sar1. Depletion of KIAA0310p mildly caused disorganization of ER exit sites and delayed protein transport from the ER, suggesting its implication in membrane traffic out of the ER. Overexpression of KIAA0310p affected ER exit sites in a manner different from that of p125. Binding experiments suggested that KIAA0310p interacts with both the inner and the outermost layer coat complexes, whereas p125 binds principally to the inner layer complex. Our results suggest that KIAA0310p, a mammalian homologue of yeast Sec16, builds up ER exit sites in cooperation with p125 and plays a role in membrane traffic from the ER.     

Cargo Selection by the COPII Budding Machinery during Export from the ER

Cargo is selectively exported from the ER in COPII vesicles. To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver cytosol and then analyzed their role in cargo selection and ER export. The purified mammalian Sec23–24 complex is composed of an 85-kD (Sec23) protein and a 120-kD (Sec24) protein. Although the Sec23–24 complex or the monomeric Sec23 subunit were found to be the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the mammalian Sec13–31 complex is required to complete budding. To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)–tagged Sar1 or GST– Sec23 to ER microsomes. Subsequently, we solubilized and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo. We find that activated Sar1 in combination with either Sec23 or the Sec23–24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein complex that excludes ER resident proteins. Supplementing these minimal cargo recruitment conditions with the mammalian Sec13–31 complex leads to export of the selected cargo into COPII vesicles. The ability of cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the ER membrane before budding and establishes the role of GTPase-dependent coat recruitment in cargo selection.     

An acidic sequence of a putative yeast Golgi membrane protein binds COPII and facilitates ER export.

We previously identified Sys1p as a high copy number suppressor of Ypt6 GTPase-deficient yeast mutants that are defective in endosome-to-Golgi transport. Here, we show that Sys1p is an integral membrane protein that resides on a post-endoplasmic reticulum (ER) organelle(s). Affinity studies with detergent- solubilized yeast proteins showed that the C-terminal 53 amino acid tail of Sys1p binds effectively to the cytoplasmic Sec23p-Sec24p COPII subcomplex. This binding required a di-acidic Asp-Leu-Glu (DXE) motif, previously shown to mediate efficient ER export of the vesicular stomatitis virus glycoprotein in mammalian cells. In Sys1p, a Glu-Leu-Glu (EXE) sequence could not substitute for the (DXE) motif. Mutations of the (DXE) sequence resulted in ER retention of approximately 30% of the protein at steady state, whereas addition of the Sys1p tail to an ER-resident membrane protein led to an intracellular redistribution of the chimeric protein. Our study demonstrates for the first time that, in yeast, a di-acidic sequence motif can act as a sorting signal for cargo selection during the formation of transport vesicles at the ER by direct binding to COPII component(s).     

The Ca2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.     

Sfb2p, a yeast protein related to Sec24p, can function as a constituent of COPII coats required for vesicle budding from the endoplasmic reticulum

The COPII coat is required for vesicle budding from the endoplasmic reticulum (ER), and consists of two heterodimeric subcomplexes, Sec23p/Sec24p, Sec13p/Sec31p, and a small GTPase, Sar1p. We characterized a yeast mutant, anu1 (abnormal nuclear morphology) exhibiting proliferated ER as well as abnormal nuclear morphology at the restrictive temperature. Based on the finding that ANU1 is identical to SEC24, we confirmed a temperature-sensitive protein transport from the ER to the Golgi in anu1-1/sec24-20 cells. Overexpression of SFB2, a SEC24 homologue with 56% identity, partially suppressed not only the mutant phenotype of sec24-20 cells but also rescued the SEC24-disrupted cells. Moreover, the yeast two-hybrid assay revealed that Sfb2p, similarly to Sec24p, interacted with Sec23p. In SEC24-disrupted cells rescued by overexpression of SFB2, some cargo proteins were still retained in the ER, while most of the protein transport was restored. Together, these findings strongly suggest that Sfb2p functions as the component of COPII coats in place of Sec24p, and raise the possibility that each member of the SEC24 family of proteins participates directly and/or indirectly in cargo-recognition events with its own cargo specificity at forming ER-derived vesicles.     

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.     

Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export

The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER). Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export. PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export. However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export. Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export. Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat.     

Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells

The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.     

Selective protein exit from yeast endoplasmic reticulum in absence of functional COPII coat component Sec13p

Sec13p has been thought to be an essential component of the COPII coat, required for exit of proteins from the yeast endoplasmic reticulum (ER). We show herein that normal function of Sec13p was not required for ER exit of the Hsp150 glycoprotein. Hsp150 was secreted to the medium under restrictive conditions in a sec13-1 mutant. The COPII components Sec23p and Sec31p and the GTP/GDP exchange factor Sec12p were required in functional form for secretion of Hsp150. Hsp150 leaves the ER in the absence of retrograde COPI traffic, and the responsible determinant is a peptide repeated 11 times in the middle of the Hsp150 sequence. Herein, we localized the sorting determinant for Sec13p-independent ER exit to the C-terminal domain. Sec13p-dependent invertase left the ER in the absence of normal Sec13p function, when fused to the C-terminal domain of Hsp150, demonstrating that this domain contained an active mediator of Sec13p-independent secretion. Thus, Hsp150 harbors two different signatures that regulate its ER exit. Our data show that transport vesicles lacking functional Sec13p can carry out ER-to-Golgi transport, but select only specific cargo protein(s) for ER exit.     

Silencing of Mammalian Sar1 Isoforms Reveals COPII‐Independent Protein Sorting and Transport

The Sar1 GTPase coordinates the assembly of coat protein complex‐II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER‐to‐Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII‐mediated transport in mammalian cells, we used small interfering RNA (siRNA)‐mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER‐to‐Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen‐I is inhibited. These data provide proof‐of‐principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII .     

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.     

Sit4p/PP6 regulates ER-to-Golgi traffic by controlling the dephosphorylation of COPII coat subunits

Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.     

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.     

Two mammalian Sec16 homologues have nonredundant functions in endoplasmic reticulum (ER) export and transitional ER organization

Budding yeast Sec16 is a large peripheral endoplasmic reticulum (ER) membrane protein that functions in generating COPII transport vesicles and in clustering COPII components at transitional ER (tER) sites. Sec16 interacts with multiple COPII components. Although the COPII assembly pathway is evolutionarily conserved, Sec16 homologues have not been described in higher eukaryotes. Here, we show that mammalian cells contain two distinct Sec16 homologues: a large protein that we term Sec16L and a smaller protein that we term Sec16S. These proteins localize to tER sites, and an N-terminal region of each protein is necessary and sufficient for tER localization. The Sec16L and Sec16S genes are both expressed in every tissue examined, and both proteins are required in HeLa cells for ER export and for normal tER organization. Sec16L resembles yeast Sec16 in having a C-terminal conserved domain that interacts with the COPII coat protein Sec23, but Sec16S lacks such a C-terminal conserved domain. Immunoprecipitation data indicate that Sec16L and Sec16S are each present at multiple copies in a heteromeric complex. We infer that mammalian cells have preserved and extended the function of Sec16.     

Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.