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J Biol Chem, Vol. 275, Issue 18, 13597-13604, May 5, 2000

Mammalian Homologues of Yeast Sec31p
AN UBIQUITOUSLY EXPRESSED FORM IS LOCALIZED TO ENDOPLASMIC RETICULUM (ER) EXIT SITES AND IS ESSENTIAL FOR ER-GOLGI TRANSPORT^*

Bor Luen Tang, Tao Zhang, Delphine Y. H. Low, Ee Tsin Wong, Heinrich Horstmann, and Wanjin Hong^§

From the Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The yeast coat protein II (COPII) is responsible for vesicle budding from the endoplasmic reticulum (ER). Mammalian functional homologues for all yeast COPII components, except for Sec31p, have been reported. We have cloned a mammalian cDNA whose product (Sec31A) is about 26% identical to Saccharomyces cerevisiae Sec31p. Data base searches also revealed another partial sequence encoding a polypeptide (Sec31B) that is 40% identical to Sec31A. Northern analysis revealed that Sec31A transcripts are ubiquitously and abundantly expressed, while Sec31B transcripts are particularly enriched in the testis and thymus, but present in very low levels in other tissues. Sec31A is localized to vesicular structures that scatter throughout the cell but are concentrated at the perinuclear region. The structures marked by Sec31A contain Sec13, a component of COPII that is well characterized to mark the ER exit sites. Immunoelectron microscopy revealed that Sec31A colocalizes with Sec13 in structures with extensive vesicular-tubular profiles. Antibodies raised against a C-terminal portion of Sec31A co-precipitate Sec13 and inhibit ER-Golgi transport of temperature-arrested vesicular stomatitis G protein in a semi-intact cell assay. Cytosol immunodepleted of Sec31A failed to support vesicular stomatitis G protein transport, which can be rescued by a high molecular weight fraction of the cytosol containing both Sec31A and Sec13. We conclude that Sec31A represents a functional mammalian homologue of yeast Sec31p.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our understanding of membrane transport in eukaryotic cells has benefited from combined genetic and biochemical approaches in yeast Saccharomyces cerevisiae (1, 2). For vesicle budding from the endoplasmic reticulum (ER),¹ the isolation and characterization of conditional lethal sec mutants (2, 3) and the development of in vitro ER budding and ER-Golgi transport assays (1) had allowed the subsequent molecular and biochemical characterization of the machinery (4-8) responsible for vesicle formation from the ER. Vesicle formation from yeast microsomal membranes has been reconstituted in vitro with three purified components from yeast cytosol, Sar1p, Sec23p·Sec24p complex, and Sec13p·Sec31p complex (9). Together, these proteins form the COPII coat complex (10, 11). They represent a set of coat proteins functioning in the early secretory pathway which are distinct from the COPI coat complex, first discovered in mammalian cells and have been proposed to mediate anterograde and retrograde transport in the secretory pathway (11-13).

The first mechanistic understanding of the function of various components of COPII with regard to vesicle formation came from the yeast system (14). In the initiation of COPII vesicle budding, the small GTPase Sar1p (6) is first recruited onto the ER membrane, at least in part, through a guanine nucleotide exchange reaction catalyzed by the ER membrane protein Sec12p (6). Sar1p on the membrane then recruits the Sec23p·Sec24p complex (15). Sec23p is a GTPase-activating protein of Sar1p (16). The resulting Sar1p-Sec23p·Sec24p complex appears to be important for cargo binding (14). Although tightly associated with Sec23p in the cytosol, Sec24p apparently does not influence the GTPase-activating protein activity of Sec23p and its exact function is unclear (15). Subsequent recruitment of the WD-40 repeat containing (17) Sec13p·Sec31p complex leads to vesicle formation, although the exact mechanistic function of the Sec13p·Sec31p complex in vesicle budding is unclear (11). These COPII components are sufficient to drive vesicle formation from chemically defined liposomes (18, 19).

Most of the corresponding mammalian homologs of yeast COPII components have now been cloned, except for Sec31p. In most cases, there exist multiple mammalian forms of a corresponding COPII component in yeast. There are two isoforms of mammalian Sar1 (20) and Sec23 (21). We and others have recently reported the cloning of four isoforms of mammalian Sec24 (22-24). A single form of mammalian Sec13 have been reported so far (25), although there exist yeast proteins homologous to Sec13p (28). We now report the existence of two mammalian proteins that are homologous to yeast Sec31p, one (Sec31A) that is abundantly and ubiquitously expressed while the other (Sec31B) is enriched in the testis and thymus. The morphological, biochemical, and functional characterization of Sec31A suggest that it is indeed a component of mammalian COPII involved in ER-Golgi transport.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Clones and Constructs-- Data base searches were performed with the various Basic Local Alignment Search Tools (BLAST) algorithms (27, 28) available at the National Center for Biotechnology (NCBI) World Wide Web server. Expressed sequence tag (EST) clones were generated by the Washington University-MERCK EST projects, and were obtained from the IMAGE consortium via Research Genetics Inc. The Sec31B partial clone was obtained from the Resource Center, German Human Genome Project, German Cancer Research Center (DKFZ) (designated DKFZp434M183). Library screening, cloning, and DNA sequencing were performed using standard methods as described (29). The EcoRI-NotI insert of an EST clone (GenBank accession number AA423899) was ³²P-labeled and used as a probe to screen a human pancreas UniZap cDNA library (Strategene) by DNA in situ hybridization. Positive clones with insert sizes larger than 3 kilobase were sequenced.

Polymerase chain reactions were carried out using high fidelity Pfu polymerase from Strategene. Fragments of the cDNA obtained were generated by polymerase chain reaction and cloned into pGEX-4T vector (Amersham Pharmacia Biotech) for the production of fusion proteins in bacteria.

Northern blot analyses were performed using human multiple tissue Northern (MTN) blots from CLONTECH (Palo Alto, CA). A full-length cDNA is used as a probe for Sec31A. For Sec31B, a fragment of about 500 base pairs corresponding to a non-homologous region of Sec31B to Sec31A was generated by polymerase chain reaction and used as a probe.

Antibodies-- Polyclonal antibody against mammalian Sec13 has been described previously (8). Antiserum against human Sec23A is kindly provided by Dr Jean-Pierre Paccaud (University of Geneva Medical Center, Switzerland) (21). Polyclonal antibodies against Sec31A in this study were obtained by immunization of mice and rabbits with a soluble glutathione S-transferase fusion construct corresponding to the C-terminal 180 amino acids of the Sec31 coding region.

Immunofluorescence Microscopy-- Cells were maintained in RPMI medium supplemented with 10% fetal bovine serum. Immunofluorescence microscopy was performed as described previously (8, 25). Cells plated on coverslips were fixed with 4% paraformaldehyde followed by sequential incubation with the primary antibodies and fluorescein isothiocyanate or rhodamine-conjugated secondary antibodies. Fluorescence labeling was visualized using an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY) with epifluorescence optics or MRC1024 (Bio-Rad) confocal laser optics.

Electron Microscopy-- Cryosectioning and immunogold labeling for electron microscopy were performed as described previously (8, 30, 31).

ER-Golgi Transport Assay-- The ER to Golgi transport assay using semi-intact cells was performed as described previously (8, 32-35). Briefly, normal rat kidney cells were grown on 10-cm dishes to confluence and infected with the temperature-sensitive strain of the vesicular stomatitis virus, VSVts045 at 32 °C for 3-4 h. The cells were then pulse-labeled with [³⁵S]methionine (100 µCi/ml) at the restrictive temperature (40 °C) for 10 min and perforated on ice by hypotonic swelling and scraping. These semi-intact cells were then incubated in a complete assay mixture of 40 µl containing 25 mM HEPES-KOH, pH 7.2, 90 mM potassium acetate, 2.5 mM magnesium acetate, 5 mM EGTA, 1.8 mM calcium chloride, 1 mM ATP, 5 mM creatine phosphate, 0.2 IU of rabbit muscle creatine phosphokinase, 25 µg of rat liver cytosol, and 5 µl (25-30 µg) of protein for 1-2 × 10⁵) of semi-intact cells. For a standard assay, samples were incubated for 90 min at 32 °C and transport terminated by transferring to ice. The membranes were collected by a brief spin and solubilized in 60 µl of 0.2% SDS, 50 mM sodium citrate, pH 5.5. After boiling for 5 min, the samples were digested overnight at 37 °C in the presence of 2.5 units of endoglycosidase H (endo H), and the reaction was terminated by adding 5× concentrated gel sample buffer. The samples were analyzed on 7.5% SDS-polyacrylamide gels and quantified using the PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For antibody inhibition of transport assay, antibodies were added into the complete assay mixture and incubated on ice for 60 min to allow the antibodies to first diffuse completely into the semi-intact cells.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Molecular Cloning of Sec31-- Data base searches using the yeast Sec31p sequence (7, 36) revealed several human EST clones encoding polypeptides with significant homology to Sec31p outside the WD-40 repeat region. We screened a human pancreas cDNA library using the insert from one of these ESTs (GenBank number AA423899) as a probe. DNA sequencing of positive clones revealed an open reading frame of 1218 amino acids. Alignment of the amino acid sequences of this mammalian protein with Sec31p is shown in Fig. 1A. It shares 25.8% identity with Sec31p, with homology throughout the entire coding region. Five WD-40 or WD-40-like repeats are present at the N terminus. There is a region enriched in proline residues in the C-terminal half. These features are similar to the domain profile of Sec31p. Although the homology between this protein and Sec31p is not as high as those found between Sar1p as well as Sec23p and their mammalian homologues (about 50%), the domain similarities suggest that this protein is a candidate for a mammalian homologue of Sec31p. We have therefore tentatively named this protein Sec31A.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   A, alignment of the amino acid sequences of Sec31A with the yeast Sec31p (MegAlign program of DNASTAR). Identical residues of these two proteins are shaded. B, table indicating the percentage of similarity and divergence of Sec31p homologues in several species aligned by the MegAlign program of DNASTAR. Shown here are the S. cerevisiae Sec31p (U15219), a Schizosaccharomyces pombe homologue (AB004537), and a C. elegans homologue (CE16320). Also in the data base but not shown here are a full-length human cDNA KIAA0905 (AB020712) deposited by the Kazusa DNA Research Institute and two alternatively spliced human cDNA (AB018358 and AB018359) deposited by M. Shibata (Medical and Biological Laboratories Co. Ltd., Nagano, Japan). There is also a rat orthologue that is about 90% homologous to the human Sec31A (Shugrue C. A. et al., Yale University School of Medicine (40)) and a partial human cDNA clone (DKFZp434M183, Wambutt et al., German Cancer Research Center) encoding a polypeptide (Sec31B) sharing 40% amino acid identity with Sec31A. C, domain structure of Sec31A protein. Sec31A, like its yeast counterparts, have a N-terminal region consisting of WD-40 repeats. There is a proline-rich region at the C-terminal half. Between these two regions, there is an intervening region without any distinct functional domain structure.

Data base search using the Sec31A sequence also revealed homology with a S. pombe and a Caenorhabditis elegans protein (Fig. 1B). These proteins also contained a region of WD-40 repeats at the N terminus and a stretch of proline-rich sequence in the C-terminal half. The most recent data base search also revealed identical full-length human clones, an alternatively spliced product, and a rat orthologue cDNA. Most interestingly, we have also identified a partial human cDNA (DKFZp434M183) encoding a 915-residue polypeptide (tentatively named Sec31B) that is about 40% identical to the C-terminal two-thirds of Sec31A. DKFZp434M183 was cloned from testis and subsequently made available to us by the Resource Center, German Human Genome Project, German Cancer Research Center.

Northern blot analysis for Sec31A revealed a transcript of about 4 kilobases in length that is abundantly and ubiquitously expressed in all tissues examined (Fig. 2). Ubiquitous expression is indeed a feature expected of an essential molecule like Sec31. We have also investigated the expression pattern of Sec31B using a polymerase chain reaction-generated fragment to a region that bears no homology to Sec31A. The Sec31B transcript is about 6.5 kilobases and is present at very low levels in most tissues compared with Sec31A. Further analysis with a different tissue panel showed that the Sec31B transcripts are particularly enriched in the thymus and testis. This finding explains our failure in finding other ESTs (except those from the same testis library) corresponding to Sec31B.

View larger version (57K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis of Sec31A and Sec31B.

Subcellular Localization of Mammalian Sec31-- Sec31A bears a relatively low homology to yeast Sec31p, while the homology of mammalian Sar1 and Sec23 to their respective yeast counterparts is much higher. This relatively weak homology brings into question as to whether Sec31A is a functional homologue of Sec31p. To facilitate our morphological, biochemical, and functional characterization of Sec31A, antibodies were raised in both mice and rabbits using a recombinant fragment representing the C-terminal portion Sec31A. Both mouse (not shown) and rabbit antisera detected a protein of about 150 kDa in all primate and rodent cells examined (Fig. 3A), which is in agreement with the size of the in vitro translated product. Indirect immunofluorescence revealed that Sec31A is associated with vesicular structures that are concentrated at the perinuclear region but are also found scattered at the cell periphery (Fig. 3B).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   A, affinity-purified rabbit antibodies against Sec31 recognized a major polypeptide of about 150 kDa, which is in agreement with the size of the [35S]methionine labeled in vitro translation product (arrow). The mouse antiserum recognized a protein of the same size (not shown). The lower molecular size polypeptides in the translation mixture are probably generated by internal initiation. It is not clear whether the lower band of about 55 kDa detected by the Sec31A antibodies correspond to a cross-reacting protein or an endoproteolytic product of Sec31A. It is also not clear whether the doublet bands in Chinese hamster ovary (CHO) cell lysates represents alternatively spliced forms. B, localization of the Sec31A in cells of different species by indirect immunofluorescence microscopy. Chinese hamster ovary (hamster), HeLa (human), or Vero (simian) cells were fixed with 4% paraformaldehyde and incubated with mouse antiserum against Sec31A, followed by incubation with fluorescein isothiocyanate-labeled anti-mouse IgG. Bar, 10 µm.

The staining pattern of Sec31A is highly reminiscent of that for mammalian Sar1 (20), Sec13 (8), Sec23A (22), and Sec24 (22), which is characteristic of ER exit sites (37). Indeed, double labeling using mouse antiserum against Sec31A and rabbit polyclonal antibodies against Sec13 showed that Sec31A colocalized well with Sec13 (Fig. 4A). Colocalization of Sec31A with an established component of mammalian COPII (Sec13) strongly supports the notion that Sec31A is also a COPII component.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   A, colocalization of Sec31A with Sec13. Normal rat kidney cells were fixed with 4% paraformaldehyde and incubated with mouse antiserum against Sec31A and rabbit polyclonal antibodies against Sec13, followed by incubation with fluorescein isothiocyanate-labeled anti-mouse IgG and rhodamine-labeled anti-rabbit IgG. The upper panels are at a lower magnification, and the lower panels represent a single cell at a higher magnification. Bar, 10 µm. B, GTPS-dependent recruitment of Sec31A onto membrane structures in permeabilized cells. Cells were permeabilized with 20 µg of digitonin in 25 mM Hepes, pH 7.3, and 125 mM potassium acetate followed by extensive washings with the same buffer. Rat liver cytosol was then added to the cells with and without 1 µM GTPS and incubated at 37 °C for 30 min. The cells were than washed, fixed with 4% paraformaldehyde, and incubated with mouse antiserum against Sec31A followed by incubation with fluorescein isothiocyanate-labeled anti-mouse IgG. Bar, 10 µm.

Coat components in both yeast and mammalian cells are recruited onto the membrane in a GTP-dependent manner. In the cases of COPI and COPII, component recruitment is initiated, respectively, by the binding and activation of the small GTPases Arf and Sar1 by their respective guanine nucleotide exchange proteins on the membrane. The localization of Sec31A on Sec13 positive membrane structures prompted us to examine if its membrane recruitment is GTP-dependent. The membrane labeling is lost or greatly diminished when cells are permeabilized with digitonin and washed extensively (Fig. 4B). This indicates that Sec31A is not tightly associated with the membrane. The very prominent localization of the protein on the membrane under steady state conditions in vivo is probably dependent on constant recruitment from the cytosol. When the permeabilized and washed cells are incubated with cytosol, binding of the protein to specific membrane structures can be observed, suggesting that there is a specific recruitment process rather than nonspecific binding. The signal observed, however, is weak compared with that in normal, unpermeabilized cells. The level of membrane association of Sec31A is greatly enhanced, approximating that in normal cells, when 0.1 µM of the nonhydrolysable GTP analog GTPS is added to the incubation. These results indicate that recruitment of Sec31A onto membrane structures marked by Sec13 is GTP-dependent.

We further examined the subcellular localization of Sec31A at the ultrastructural level. Double immunogold labeling of Sec31A and Sec13 revealed that they are both enriched in structures with a vesiculated membrane profile around the Golgi apparatus (Fig. 5). This is consistent with the results obtained at the light microscopy level. Examination at a higher magnification showed that the structures in which both Sec13 and Sec31A are concentrated appear to consist of buds, vesicles, and tubules. These structures are likely to be the sites of COPII vesicle budding from the ER.

View larger version (204K): [in this window] [in a new window]   Fig. 5.   Colocalization of Sec31A and Sec13 at the ultrastructural level. Vesiculated profiles enriched in both Sec31A (14-nm gold particles) and Sec13 (10-nm gold particles) are frequently found around the Golgi apparatus (labeled G) (a). These structures appear to be enriched in buds, vesicles, and tubules (b-d). Panel b is a magnified view of the Sec13/Sec31A containing structures shown in panel a. Bar, 0.1 µm.

Sec31A Exists as a High Molecular Weight Complex with Sec13-- The colocalization of Sec31A with Sec13 led us to examine whether the two proteins interact in vivo. As shown in Fig. 6, antibodies against Sec31A co-precipitates the 36-kDa Sec13. In the reciprocal experiment, antibodies against Sec13 (8) co-precipitates the 150-kDa Sec31A. This interaction between Sec31A and Sec13 manifested by co-immunoprecipitation is specific because Sec23, another COPII component in the cytosol, does not co-precipitate with either Sec31A or Sec13 under the same conditions.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Sec31A interacts physically with Sec13 but not Sec23 in the cytosol. Rat liver cytosol was immunoprecipitated with rabbit polyclonal antibody against Sec31A (A and D) or Sec13 (B and C). The immunoprecipitates were eluted in SDS-PAGE sample buffer and subjected to SDS-PAGE together with 1/25th of the cytosol used in each immunoprecipitation. The proteins were transferred to nitrocellulose and immunoblotted using affinity-purified rabbit polyclonal antibodies against Sec13 (A), Sec31A (B), or antiserum against Sec23A (C and D) (21). The molecular sizes of markers used (in kDa) were indicated on the left of each panel, and arrows on the right point to the detected bands of interest as well as the immunoglobulin heavy chain (IgH). In panel E, rat liver cytosol was fractionated by gel filtration on a Superose 6 column. The fractions were precipitated by 10% trichloroacetic acid and equivalent amounts of each were subjected to SDS-PAGE and immunoblotting using either the mouse antiserum against Sec31A or rabbit polyclonal antibodies against Sec13. Fractions at which molecular size markers peaked are indicated.

We further examined the Sec13·Sec31A complex by gel filtration. When rat liver cytosol was subjected to gel filtration in a Superose 6 medium (Amersham Pharmacia Biotech), the majority of Sec13 as well as Sec31A were fractionated to high molecular weight fractions of about 600-700 kDa (Fig. 6E). A very small fraction of each is also found in low molecular weight fractions with sizes corresponding to monomeric forms of the respective proteins.

In view of the fact that Sec13 and Sec31A exists essentially in a complex in the cytosol, we have estimated their interaction stoichiometry based on the incorporation of [³⁵S]methionine into the polypeptides. HeLa cells starved in medium without methionine was subsequently incubated for 5 h in medium containing 0.25 mCi/ml [³⁵S]methionine. Lysates of the cells were subjected to immunoprecipitation with antibody against Sec13 and antibody against Sec31A. When resolved on a SDS-PAGE gel, both immunoprecipitates contain bands corresponding to Sec13 (36 kDa) and Sec31A (150 kDa) in similar ratios, and subsequent quantitation of the bands by scintillation counting revealed an approximate 1:1 stoichiometry of the two proteins.

Sec31 Is Essential for ER-Golgi Transport-- The subcellular colocalization and biochemical interaction between the Sec31A and Sec13 strongly suggest that Sec31A may represent the functional counterpart of yeast Sec31p. We have therefore sought further functional evidence for the involvement of Sec31A in ER-Golgi transport.

We used a permeabilized cell system monitoring the transport of temperature-synchronized vesicular stomatitis virus (ts045 strain) G protein (VSVG) (8, 32, 34-35). As shown in Fig. 7A, the fusion protein between the C-terminal 180 amino acids of Sec31A and GST (GST-Sec31A) inhibited VSVG transport, as does GST-Sec22B (34), whereas GST itself had no effect. Antibodies raised against GST-Sec31A also inhibited transport, as do antibodies against Bet1 (35) and Sec22B (34), whereas control rabbit IgG has no effect. The inhibitory activity of Sec31A antibodies could be inactivated by heat denaturation (not shown). The fact that antibodies against Sec31A inhibit ER-Golgi transport as do antibodies against SNARE molecules like Bet1 (35), Sec22B (34), and GS28 (38) strongly suggest that Sec31A is involved in ER-Golgi transport.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Sec31A is essential for ER-Golgi transport. A, transport assay mixtures were supplemented with GST, GST-Sec31A, GST-Sec22B, rabbit IgG, or antibodies against Sec31A, Bet1 (35), or Sec22B (34). Transport reactions and processing were carried out as described under "Materials and Methods." The lower band corresponds to the endo H-sensitive ER form of VSVG, and the upper band corresponds to the endo H-resistant Golgi form. The ratio of the intensities of the upper band and lower band is a measure of transport of VSVG from the ER to the Golgi. The respective values of which are plotted on the graph. B, essential role of cytosolic Sec31A in ER-Golgi transport. Immunodepletion of rat liver cytosol was carried out by incubating cytosol with protein A-Sepharose beads coated with Sec31A antibodies or rabbit IgG (mock depletion). The extends of the depletions were determined by quantitative immunoblot with Sec31A antibodies (upper panel). Transport assay mixtures were supplemented with increasing amounts of Sec31A-depleted or mock-depleted cytosol, and the resulting ratios of the Golgi versus the ER form were plotted on the graph below (filled squares, mock-depleted cytosol; filled circles, Sec31A-depleted cytosol). C, rat liver cytosol was fractionated by gel filtration on a Superose 6 column. The high molecular size peak fractions containing Sec31A as determined by immunoblotting were than used in transport assays (HMF-high molecular size fraction; Dep. Cytosol-immunodepleted cytosol). The amounts of cytosol and HMF used to supplement the Dep. cytosol contained equivalent amounts of Sec31A, as determined by quantitative immunoblot (not shown).

We next examine if the presence of Sec31A in the cytosol is essential for ER-Golgi transport. To do this, we immunodepleted Sec31A from rat liver cytosol and compared the efficacy of the Sec31A-depleted cytosol in supporting VSVG transport to cytosol mock-depleted with rabbit IgG. As shown in Fig. 7B, Sec31A-depleted cytosol was unable to support VSVG transport to any significant level even at high concentrations.

Sec31A, in complex with Sec13, is likely to be the missing ingredient from the Sec31A-depleted cytosol that resulted in lost of transport activity. To prove this, we checked if the high molecular weight fraction enriched in Sec31A and Sec13 could restore the transport activity of Sec31A-depleted cytosol. As shown in Fig. 7C, the Sec31A depleted-cytosol and the high molecular size fraction by themselves do not support transport. However, addition of the high molecular size fraction to Sec31A-depleted cytosol efficiently restores its transport activity, to a level comparable to that of normal cytosol. The above results suggest that Sec31A is essential for the ER-Golgi transport of VSVG.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mammalian Homologues of Yeast Sec31p-- Sec31p was first discovered as a 150-kDa protein interacting with Sec13p in the yeast cytosol (39), and was shown biochemically to be essential for vesicle formation from the ER (9, 39). The SEC31 gene was subsequently cloned by complementation of the temperature sensitive Sec31-1 allele (7). SEC31 was also cloned separately as WEB1, allele-specific mutations of which lead to growth dependence on the adenovirus E1A protein (36).

In this report, we described two mammalian proteins that are homologous to Sec31p. Sec31A is abundantly and ubiquitously expressed, while Sec31B is enriched in the thymus and testis. Although sharing a mere 26% homology to yeast Sec31p, three lines of evidence suggest that Sec31A is a functional counterpart of Sec31p. First, Sec31A colocalized well with Sec13. Without any recognizable hydrophobic sequences that would serve as a membrane anchor, Sec31A nevertheless showed membrane staining patterns which are very similar to that of other known mammalian COPII components (8, 20-22). This suggests that they were recruited to the same membrane structures, a fact that is further verified by the observation at the ultrastructural level that both Sec31A and Sec13 are present in vesicular-tubular structures characteristic of the ER exit sites. Moreover, like Sec13 and other COPII components, recruitment of Sec31A to membranes is GTP-dependent, and recruitment in permeabilized cells is greatly enhanced by the nonhydrolysable analog GTPS.

Second, Sec31A exists in a protein complex with Sec13. In the cytosol, antibodies against Sec31A and antibodies against Sec13 can reciprocally co-immunoprecipitate one another. On the other hand, Sec23, whose yeast counterpart Sec23p has been shown to be in a different complex (the Sec23p-Sec24p complex) in the cytosol (15), could not be co-immunoprecipitated by either antibodies. Sec31A also cofractionates with Sec13 to a high molecular weight fraction, as determined by gel filtration analysis of rat liver cytosol. It is interesting to note that the high molecular weight complex of Sec31A and Sec13 could not be formed easily in vitro, for example, by simply mixing the in vitro translation products of Sec31A and Sec13 (data not shown). The cytosolic environment may therefore aid complex formation in vivo, perhaps also with the help of chaperone proteins.

Finally, we demonstrated that Sec31A is essential for ER-Golgi transport. Both the C-terminal 180-amino acid fusion protein as well as antibodies against Sec31A inhibited ER-Golgi transport of VSVG in a semi-intact cell system. Immunodepletion of Sec31A from the cytosol impaired its ability to support transport. This impairment could be rescued by the high molecular fractions containing Sec31A and Sec13. Taken together, the evidence for Sec31A being a mammalian equivalent of yeast Sec31p is compelling.

It appears that most mammalian COPII components have more isoforms than their yeast counterparts. This is probably a reflection of the higher level of sophistication and complexity in the mammalian machinery and also perhaps a need to delegate the recruitment of specific subsets of cargo proteins to combinations of different COPII components. It is not difficult to envisage that in cells specializing in the secretion of particular proteins, there may exist cell type-specific isoforms of a general component of the transport machinery. However, it is not clear at present if the thymus and testis-enriched Sec31B functions in COPII vesicle formation. While this report is under review, Shugrue et al. (40) reported the cloning and characterization of a rat protein, p137, that bears about 90% identity to Sec31A. The rat protein is also ubiquitously expressed in rat tissues and is very likely the rat orthologue of human Sec31A.

Interaction of Sec31A with Sec13 and Other Proteins-- The coat protein complexes responsible for vesicle budding in the early secretory pathway share a similar feature in the mechanism of membrane recruitment, via the activation and membrane recruitment of a small GTPase. The components of the COPI complex exist in the cytosol as a seven-polypeptide complex (known as the coatomer) and is recruited en bloc onto the membrane via activation of Arf (41). In contrast, COPII proteins exist as smaller subcomplexes in the cytosol and are sequentially recruited onto the membrane (9, 14, 42). Although the Sec23-24 complex together with activated Sar1 are apparently important for the recruitment of cargo proteins to the budding sites (14, 42), the role of subsequent binding of Sec13·Sec31 complex is unknown. It is speculated that these proteins perhaps serve a function analogous to that of clathrin triskelons in clathrin-coated vesicles (43), in that they may cluster bound components together with cargo molecules into the shape of coated vesicles. In support of this, yeast Sec31p has been shown to interact directly with both Sec23p and Sec24p in vitro as well as in the context of the yeast two-hybrid system (44). It may also be relevant to note that the native Sec13·Sec31 complex is large, presumably multimeric, and will be well suited for the proposed function.

It has also been shown that yeast Sec31p interacts with Sec16p, a peripheral membrane protein tightly associated with the ER membrane (44). Sec16p, which also binds to Sec23p and Sec24p, is a large multidomain protein which may serve a docking function in bringing all the COPII components together (45). Although the mammalian homologue of Sec16p has not yet been cloned, its existence was recently demonstrated as a band immunologically related to Sec16p in a GST-Sec23 pull-down experiment (46). It is interesting that the domain responsible for interacting with Sec16p was mapped to the C terminus of Sec31p, as it may account for the transport inhibitory effect of the C-terminal fusion protein as well as the antibodies against this region of Sec31A. The mechanism of inhibition, however, is probably more than a simple inhibition of binding or recruitment, as the antibody does not inhibit the recruitment of the Sec13·Sec31 complex onto membranes in vitro (data not shown).

The Sec13p·Sec31p complex in the yeast cytosol appears to consist of only those two proteins (39), and we have not yet been able to identify any other proteins that may be associated with the Sec13·Sec31A complex in rat liver cytosol. It may be that interactions of other proteins with the Sec13·Sec31A complex in the cytosol are not robust enough to be detected by coprecipitation methods. However, both Sec13 and Sec31A may well interact with proteins other than Sec23 and Sec24 when recruited onto the membrane. In fact, our immunoprecipitation of [³⁵S]methionine-labeled total cell lystes with either antibodies against Sec13 or Sec31A revealed several bands with molecular sizes distinct from Sec23, Sec24, or Sar1 (data not shown).

There is a precedence for interactions of COPII components with other proteins in a Sar1-independent manner. Sec23A has been recently shown to interact directly with p125, a protein with homology to phospholipid-modifying proteins (46). Yeast Sec24p has been shown to interact specifically with the cis-Golgi syntaxin Sed5p (47). The WD-40 repeats at the N terminus of Sec31 are known motifs for protein-protein interaction. This domain of Sec31p presumably interacts with Sec13p (36), but may well bind other proteins. The C-terminal half of Sec31A contains a proline-rich region with several SH3 binding consensus sites (XPXXP) (48). As the last complex of COPII to be recruited onto membranes prior to vesicle formation, it makes sense to link this complex to regulatory mechanisms. It would not, therefore, be surprising if Sec31 interacts with other proteins regulating COPII vesicle formation.

	ACKNOWLEDGEMENT

We thank Dr Jean-Pierre Paccaud (University of Geneva Medical Center, Switzerland) for antiserum against human Sec23A.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) of Sec31A reported in this paper has been submitted to GenBank^TM/EBI Data Bank with the accession number(s) AF139184.

To whom correspondence should be addressed. Tel.: 65-874-3793; Fax: 65-779-1117; E-mail: mcbtbl@imcb.nus.edu.sg.

^§ Supported by a research grant from the Institute of Molecular and Cell Biology. To whom correspondence should be addressed. Tel.: 65-874-3762; Fax: 65-779-1117; E-mail: mcbhwj@imcb.nus.edu.sg.

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; EST, expressed tag sequence; GTPS, guanosine 5'-O-(thiotriphosphate); PAGE, polyacrylamide gel electrophoresis; VSVG, vesicular stomatitis G protein; GST, glutathione S-transferase.

	REFERENCES

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