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J. Biol. Chem., Vol. 277, Issue 24, 21955-21961, June 14, 2002

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Sec34 Is Implicated in Traffic from the Endoplasmic Reticulum to the Golgi and Exists in a Complex with GTC-90 and ldlBp^*

Eva Loh and Wanjin Hong

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

Received for publication, March 11, 2002, and in revised form, April 2, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein transport from the ER¹ to the Golgi in mammalian cells involves several key steps (1, 2). Cargo proteins and components necessary for downstream steps are first exported from the ER exit sites via the action of COPII components (3, 4). COPII vesicles and/or clusters of COPII vesicles are believed to undergo homotypic fusion to form pleiotropic transport intermediates (1, 5). The ER exit sites and transport intermediates have been collectively referred to as the intermediate compartment or the ER-Golgi intermediate compartment (2, 6). The heterotypic fusion of the transport intermediates with the cis-Golgi results in the delivery of cargo proteins to the Golgi apparatus. Many proteins originally identified in yeast have now been shown to participate in ER-to-Golgi transport in mammalian cells (2, 7), and more proteins regulating ER-to-Golgi transport in mammalian cells are expected to exist.

SEC34 and SEC35 were identified as genes whose products are necessary for protein transport from the ER to the Golgi in yeast Saccharomyces cerevisiae (8). More detailed studies of Sec34p and Sec35p have suggested that they function as components of a protein complex that acts as a tethering factor to ensure the proper docking and fusion of transport intermediates with the Golgi apparatus (9-12). In addition, the TRAPP protein complex (13) and Uso1p (14-16) have similarly been shown to function in tethering for the same transport event. Although the spatial, temporal, and mechanistic relationships among the Sec34-Sec35p complex, the TRAPP complex, and Uso1p have yet to be fully characterized, the importance of the tethering process in ensuring faithful docking and fusion of transport intermediates with the target compartment is becoming more recognized in several trafficking events (17-19). Although the mammalian homologue (Sec34) of Sec34p has been molecularly identified (20), its role in membrane traffic in mammalian cells remains to be established, and its interacting partners remain to be explored.

GTC-90 was purified as a component of a novel protein complex in mammalian cells that is required for intra-Golgi transport in vitro (21). Although the GTC-90 protein complex is known to include several other different subunits, their identities are unknown, and the functional aspects of the GTC-90 complex in other transport events remain to be examined.

Low density lipoprotein receptor is responsible for the clearance of serum low density lipoprotein particles, and diverse mutations in its gene are associated with familial hypercholesterolemia (22). Due to its importance, understanding the pathway and mechanism underlying low density lipoprotein receptor trafficking has been an active area of cell biology. Genetic approaches have been used to create and identify several mutant lines of Chinese hamster ovary cells, including ldlA-ldlI (23-25). The genes mutated in some of these mutants (such as ldlA, ldlB, lldC, ldlD, and ldlF) have been identified (26-30). Biochemical and cell biological characterizations of ldlBp and ldlCp have revealed that they are components of the same protein complex (28, 30). Both ldlBp and ldlCp are necessary for maintaining normal structure and function of the Golgi apparatus, although the precise function remains to be investigated.

Using antibodies against the recombinant Sec34 N-terminal fragment, evidence is presented to support a role for Sec34 in ER-to-Golgi transport in mammalian cells. Significantly, the observation that antibodies against Sec34 could co-immunoprecipitate GTC-90, ldlBp, and others suggests that Sec34, GTC-90, ldlBp, and ldlCp are part of the same protein complex(es) that may regulate diverse aspects of the Golgi functions, including ER-to-Golgi transport.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All cell lines were obtained from the American Type Culture Collection (Manassas, VA). Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences. Fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (IgG) and Texas Red-conjugated goat anti-mouse IgG were from Jackson ImmunoResearch. Restriction enzymes were all purchased from Roche Molecular Biochemicals. Local New Zealand White rabbits were purchased from the Sembawang Laboratory Animals Centre (Singapore). Freund's adjuvants (complete and incomplete) were from Invitrogen. The human cDNA clones KIAA1134 and KIAA1381 were kindly provided by the Kazusa DNA Research Institute. Human expressed sequence tag clone accession number AA439818 was generated by the Washington University-Merck Expressed Sequence Tag Project and made available by the IMAGE consortium via Research Genetics Inc. (Huntsville, AL). cDNA clone AI492237 was purchased from Invitrogen. AK026305 cDNA clone was provided by the NEDO (New Energy and Industrial Technology Development Organization) Human cDNA Sequencing Project. Synthetic oligonucleotides were ordered from Research Genetics (Singapore).

cDNA Cloning of Human Sec34-- Full-length human Sec34 cDNA was assembled by first obtaining a 1.8-kb fragment from cDNA clone AK026305 by digestion with XhoI and NsiI. This fragment, which contains the 5' coding region of Sec34, was ligated to an ~3.6-kb fragment obtained from cDNA clone AA439818 by digestion with XhoI and NsiI, which also includes the vector pT7T3D-Pac. An additional 1.8-kb fragment was obtained from cDNA clone AA439818 by digestion with NsiI alone. These fragments were ligated to construct an overall 4.3-kb Sec34 cDNA in pT7T3D-Pac vector.

Expression Constructs for myc Epitope-tagged Sec34, Dor1, Cod1, Cod2, ldlBp, and ldlCp Myc-Sec34-- Sec34 cDNA in pT7T3D-Pac vector was digested with XhoI and NotI, and the resulting fragments were digested with BglI to obtain an ~2.5-kb fragment that contains the entire coding region of Sec34. This fragment was blunt-ended and ligated to pDMyc-neo vector (31) pre-cut with XbaI and blunt-ended. For Myc-Cod2, primer 1 (5'-GAG-CTC-GAG-CGG-GCA-GAG-GGC-AGC-GGG-GAA-GTG) and primer 2 (5'-GGC-ATC-TGC-AAC-TTG-AGC-TCT-TAT-CTC-TAA-TTT) were used to amplify a 450-bp fragment from the KIAA1134 clone, and the resulting PCR product was digested with XhoI and SacI. This fragment was ligated to another fragment retrieved by digesting KIAA1134 with SacI and NotI, together with the pDMyc-neo vector pre-cut with XhoI and NotI. For Myc-ldlBp, primer 3 (5'-GAG-CTC-GAG-CGG-GTG-GGC-GAA-CGG-TAC) and primer 4 (5'-TAC-ACA-TGT-GGA-TCC-ATT-TCT-GCA-GC) were used to amplify an ~1-kb fragment from KIAA1381, and the resulting PCR fragment was digested with XhoI and BamHI. This was ligated to a fragment retrieved from KIAA1381 by digestion with BamHI and NotI, together with pDMyc-neo vector pre-cut with XhoI and NotI. For Myc-ldlCp, primer 5 (5'-GGC-CTC-GAG-CGG-GAG-AAA-AGT-AGG) and primer 6 (5'-GGT-CTA-GAC-GAG-AGG-CTG-CTC-TGC-TGT-TGC) were used to amplify the entire coding sequence of ldlCp from IMAGE clone AI492237 by PCR, and the resulting product was digested with XhoI and XbaI and ligated into the corresponding sites of pDMyc-neo vector. For Myc-Dor1 and Myc-Cod1, plasmids SDOR1M1 and SCOD1M4, which express Dor1 and Cod1, respectively, with triple myc tags at the C terminus were kindly provided by Dr. Sean Munro (32).

Expression and Purification of Recombinant GST Fusion Proteins-- For production of recombinant GST fusion proteins, primer 7 (5'-ATT-GGA-TCC-ATT-ATG-GCG-GAG-GCG-GCG) and primer 8 (5'-CGG-CTC-GAG-CGG-ACT-TGT-GAG-GGT-CTG-TAG-TGT-GTT) were used to amplify the coding sequence for residues 1-276 of Sec34. This PCR product was digested with BamHI and XhoI. Primer 9 (5'-CTC-CCG-GGT-CAT-CAG-TTA-CTG-AAA-AGG-GAT-CCT) and primer 10 (5'-CGG-CTC-GAG-CGG-TCC-ATG-AAG-ATC-TGC) were used to retrieve the region coding for residues 277-552 of Sec34. The PCR product was digested with SmaI and XhoI. Primer 11 (5'-CCC-GGA-TCC-CCC-ATG-TGG-TAT-CCT-ACG-GTT-CGA-AGA) and primer 12 (5'-CGG-CTC-GAG-CGG-TTT-AGA-AAC-TGA-CAG-CAG-AAG) were used to amplify the sequence coding for residues 553-828 of Sec34. This PCR product was digested with BamHI and XhoI. The bacterial expression vector pGEX-4T-1 (Amersham Biosciences) was digested accordingly and ligated with these PCR fragments. The ligated plasmids were transformed into DH5 cells, and ampicillin-resistant colonies expressing the GST fusion proteins were screened as described previously (33). Purification of GST fusion proteins was performed as described previously (34).

Preparation and Affinity Purification of Sec34 Antibodies-- 400 µg of GST-Sec34/F1 was emulsified with Freund's complete adjuvant and injected subcutaneously into two local New Zealand White rabbits. Booster injections with the same amount of antigen in Freund's incomplete adjuvant were administered every 2 weeks. The rabbits were bled 10 days after the second and subsequent boosters. For affinity purification, the antiserum was first diluted with an equal volume of PBS and then incubated for 2 h at 4 °C with GST coupled to cyanogen bromide-activated Sepharose 4B (Amersham Biosciences) to remove antibodies against GST. The flow-through was then incubated overnight at 4 °C with GST-Sec34/F1-coupled beads. The beads were washed extensively, and Sec34-specific antibodies were eluted with low pH elution buffer as described previously (34).

Immunoblot Analysis-- Proteins were separated by SDS-PAGE and electrotransferred onto Hybond C+ nitrocellulose. The blots were then incubated for 1 h at 37 °C in blocking buffer (5% skim milk and 5% fetal bovine serum in PBS containing 0.05% Tween 20). The blots were incubated in blocking buffer containing primary antibodies for 1 h at room temperature, followed by three washes (5 min each) with PBS containing 0.05% Tween 20. The blots were then incubated with either goat anti-rabbit or anti-mouse antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch). After three washes in PBS containing 0.05% Tween 20, SuperSignal West Pico Chemiluminescence Substrate (Pierce) was added, and the blots were processed according to the manufacturer's protocol. For the blocking experiment shown in Fig 2A, 50 µg of proteins extracted from Golgi-enriched membranes or cytosol was electrophoresed and transferred to a filter. After blocking, the filter was immunoblotted with 2 µg of affinity-purified Sec34 antibodies or with 2 µg of Sec34 antibodies preincubated with 20 µg of recombinant GST-Sec34/F1, GST-Sec34/F2, or GST-Sec34/F3.

Immunofluorescence Microscopy-- Cells were grown on coverslips overnight to 50-80% confluence, rinsed twice with phosphate-buffered saline with 1 mM CaCl₂ and 1 mM MgCl₂, and processed as described previously (34, 35). Sec34 antibodies (5-10 µg/ml) in fluorescence dilution buffer (PBSCM with 5% normal goat serum, 5% fetal bovine serum, and 2% bovine serum albumin, pH 7.6) were used. 0.5 µg of GST-Sec34/F1 was used to neutralize the antibodies.

In Vitro ER-to-Golgi Transport Using Semi-intact Cells-- The ER-to-Golgi transport assay using semi-intact cells was an assay modified according from a previously reported procedure (7, 36) and was performed as follows. Briefly, normal rat kidney cells grown on 10-cm Petri dishes as a confluent monolayer were infected with a temperature-sensitive strain of the vesicular stomatitis virus, VSVts045, at 32 °C for 1 h and then at the restrictive temperature of 40 °C for another 2 h. The cells were then subjected to perforation on ice by hypotonic swelling and scraping. These semi-intact cells were then incubated in a complete assay mixture (40 µl) containing 25 mM Hepes-KOH, pH 7.2, 90 mM KOAc, 2.5 mM MgOAc, 5 mM EGTA, 1.8 mM CaCl_2, 1 mM ATP, 5 mM creatine phosphate, 0.2 IU of rabbit muscle creatine phosphokinase, 25 µg of cytosol, and 5 µl (25-30 µg of protein; 1-2 × 10⁵) of semi intact cells. Additional reagents were added as indicated. For a standard assay, samples were incubated for 90 min at 32 °C, and transport was terminated by transferring samples to ice. The membranes were collected by a brief spin and solubilized in 20 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, and the reaction was terminated by adding 6× concentrated gel sample buffer. The samples were separated on 7.5% SDS-polyacrylamide gels and transferred to a nitrocellulose filter. Immunoblot analysis was performed with anti-vesicular stomatitis virus antibodies (Roche Molecular Biochemicals). For antibody inhibition of transport assay, Sec34 antibodies were added into the complete assay mixture and incubated on ice for 60 min to allow diffusion of antibodies into the semi-intact cells.

Large-scale Immunoprecipitation-- 300 µl of protein A-Sepharose CL-4B (Amersham Biosciences) was washed in PBS and used to bind 500 µg of rabbit anti-goat IgG (Pierce) or 500 µg of anti-Sec34 antibodies separately for 2 h at 4 °C. The antibodies were cross-linked to the beads with 50 mM dimethyl pimelidate in 0.2 M sodium borate, pH 9, overnight at 4 °C and then washed in 0.2 M sodium borate, pH 9, and incubated with 0.2 M ethanolamine, pH 8, for 2 h at room temperature. The beads were washed with PBS and finally in 0.5% TX-100 in gradient buffer (20 mM Hepes, pH 7.3, 100 mM KCl, and 2 mM EDTA). 50 mg of rat liver cytosol was first incubated with the beads bound with control rabbit IgG in gradient buffer with 0.5% TX-100 for 2 h at 4 °C. The supernatant was then incubated with the beads bound to Sec34 antibodies overnight at 4 °C. The beads were then washed three times with gradient buffer containing 0.5% TX-100 and then finally with gradient buffer without TX-100.

Transfection and Analytic Immunoprecipitation-- 293T cells were grown on 60-mm dishes to 40-60% confluence. 1 µg of myc-tagged ldlBp, ldlCp, Dor1, Cod1, and Cod2 DNA was used for transfection with Effectene Transfection Reagent, according to the manufacturer's instructions (Qiagen). The next day, the cells were washed twice with PBSCM. 150 µl of PBS containing 1% TX-100 and complete EDTA-free protease inhibitor mixture (Roche Diagnostics) was added, and the cells were then scraped with a cell scraper. The cells were rotated at 4 °C for 15 min and then spun down at 4000 rpm for 10 min. Sec34 antibodies (5 µg) were added to the supernatant and incubated at 4 °C for 2 h. Next, protein A-Sepharose CL-4B beads were added and left to bind overnight at 4 °C. The beads were then washed three times with PBS and 1% TX-100, washed with PBS and 0.2% TX-100, and finally washed with PBS and analyzed by SDS-PAGE. Immunoblot analysis was performed with anti-myc monoclonal antibodies.

In Vitro Translation and Binding Experiment-- TNT T7 Quick Master Mix (Promega) was used for in vitro translation according to manufacturer's protocol. In a 100-µl reaction, myc-tagged Sec34 and each of the myc-tagged ldlBp, ldlCp, and Cod2 were co-translated for 2 h at 30 °C. 20 µl of each reaction was used for co-immunoprecipitation with 10 µg of anti-myc antibodies (rabbit polyclonal IgG) (Upstate Biotechnology) or 10 µg of anti-Sec34 antibodies bound to protein A-Sepharose beads in gradient buffer with 0.1% TX-100. After 1 h at room temperature, the beads were washed five times with gradient buffer with 0.1% TX-100 and analyzed by SDS-PAGE and autoradiography together with 2 µg of in vitro-translated product (10% starting material).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of Antibodies against Sec34-- Due to the established role of Sec34p in ER-to-Golgi transport in yeast and our general interest in ER-to-Golgi transport in mammalian cells, we used the amino acid sequence of Sec34p to search for its putative mammalian counterpart by BLAST searches (37). Several cDNA sequences encoding polypeptides homologous to various regions of Sec34p were uncovered. The complete coding region of human Sec34 was assembled from various cDNAs and confirmed by DNA sequencing (GenBank^TM accession no. AF332595). During the course of our work, the molecular identification of human Sec34 was independently reported (20). Examination of the deduced 828 amino acid sequences suggests that Sec34 could be roughly divided into two regions (Fig. 1A). The N-terminal one-third is highly homologous to counterparts from other species such as yeast, fly, and worm and has the potential (particularly residues 126-211) to form coiled-coil structures. The C-terminal two-thirds is homologous to EEA1 (Fig. 1B), a well-established tethering factor that regulates endosome fusion (38). The structural relatedness of Sec34 to a well-defined tethering protein provides some structural evidence for Sec34 to function as a tethering factor. To define the functional and biochemical aspects of Sec34, we expressed recombinant Sec34 in three separate fragments (residues 1-276, Sec34/F1; residues 277-552, Sec34/F2; and residues 553-828, Sec34/F3) fused to GST (Fig. 1A). GST-Sec34/F1 was used to raise antibodies against Sec34. As shown in Fig. 1C, affinity-purified Sec34 antibodies recognized a polypeptide of about 93 kDa in both membrane (lane 1) and cytosol (lane 5) fractions derived from rat liver. Detection of this polypeptide by the antibodies was abolished by preincubation of the antibodies with GST- Sec34/F1 (Fig. 1C, lanes 2 and 6) but not with GST-Sec34/F2 (lanes 3 and 7) or GST-Sec34/F3 (lanes 4 and 8), suggesting that the antibodies are specific for Sec34 and that Sec34 is present in both cytosolic and membrane fractions. Using these antibodies in immunofluorescence microscopy, Sec34 was seen to be enriched in the Golgi apparatus (Fig. 1D, a) marked by the Golgi SNARE GS28 (Fig. 1D, b) (39, 40) in HeLa cells. Furthermore, the Golgi labeling of Sec34 (Fig. 1D, d) but not GS28 (Fig. 1D, e) was abolished by preincubation of the antibodies with GST-Sec34/F1, further confirming the specificity of the antibodies. These results are similar to those reported previously (20) and suggest that our antibodies are specific for Sec34.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Characterization of Sec34 antibodies. A, domain organization of Sec34. The 828-residue Sec34 can be divided into the N-terminal one-third that is conserved among proteins from various species and has the potential to form coiled-coil structures; whereas the C-terminal two-thirds is homologous to EEA1. The regions from which various GST fusion proteins were derived are indicated. B, the C-terminal two-thirds of Sec34 is homologous to EEA1. Residues 301-810 of Sec34 were aligned with residues 610-1109 of EEA1. Identical residues are shown in red, whereas conserved residues are shown in pink. C, antibodies raised against GST-Sec34/F1 specifically recognize a 93-kDa protein. The 50 µg of Golgi-enriched membrane fractions (lanes 1-4) and 50 µg of total cytosol (lanes 5-8) derived from rat liver were resolved by SDS-PAGE and transferred to filters. The filters were incubated with either Sec34 antibodies alone (lanes 1 and 5) or in the presence of GST-Sec34/F1 (lanes 2 and 6), GST-Sec34/F2 (lanes 3 and 7), or GST-Sec34/F3 (lanes 4 and 8). D, anti-Sec34 antibodies label the Golgi apparatus. HeLa cells were fixed, permeabilized, and double-labeled with Sec34 antibodies (a and d) and monoclonal antibodies against Golgi SNARE GS28 (b and e). The Golgi labeling of Sec34 (d) but not GS28 (e) was abolished by prior incubation of the antibodies with GST-Sec34/F1. The merged images are shown in D, c and f. Bar, 10 µm.

A Role for Sec34 in ER-to-Golgi Transport-- Because evidence for a functional role for Sec34 in ER-to-Golgi transport in mammalian cells is lacking, we investigated the potential transport function of Sec34 by using a modified semi-intact cell assay that reconstitutes protein transport from the ER to the Golgi (see "Experimental Procedures" for details). NRK cells were infected with a temperature-sensitive mutant vesicular stomatitis virus (VSVts045) at 32 °C for 1 h followed by incubation at 40 °C for 2 h to accumulate its envelope protein (VSVG) in the ER. Cells were then permeabilized by scraping in hypotonic buffer and used to reconstitute transport of VSVG by supplementing them with exogenous rat liver cytosol and an ATP-regenerating system at 32 °C. Transport of VSVG to the Golgi was measured by monitoring the conversion of its endoglycosidase H-sensitive glycans to endoglycosidase H-resistant forms of the entire population of ER-arrested VSVG molecules as revealed by immunoblot analysis. There are two advantages in this modified assay as compared with the original protocol (7). The first is that no radioactive materials were used. The other is that instead of measuring a small fraction (the radiolabeled pool) of total VSVG, this assay measures the synchronized transport to the Golgi of almost all VSVG molecules accumulated in the ER. As shown in Fig. 2, VSVG remained as the endoglycosidase H-sensitive ER form when the transport reaction was performed on ice (Fig. 2A, lane 1; Fig. 2B, lane 1) or in the absence of cytosol (Fig. 2A, lane 2; Fig. 2B, lane 2). Between 60% and 90% of total VSVG was converted into the endoglycosidase H-resistant form when the transport assay was conducted in the presence of complete mixture at 32 °C (Fig. 2A, lane 3; Fig. 2B, lane 3). Addition of antibodies against GST (Fig. 2A, lanes 4-6) did not inhibit this transport, whereas the addition of antibodies against Sec34 (Fig. 2A, lanes 7-10) inhibited the transport in a dose-dependent manner. Clear inhibition was observed in the presence of 3 µg of anti-Sec34 antibodies, and the transport was almost completely inhibited by 7 µg of antibodies (Fig. 2A, lane 10). The inhibition by Sec34 antibodies is specific because it could be neutralized by a noninhibitory amount of GST-Sec34/F1 (Fig. 2B, lanes 5 and 7), but not by GST (Fig. 2B, lanes 4 and 6). These results suggest that Sec34 is important for ER-to-Golgi transport in mammalian cells.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Sec34 antibodies specifically inhibit ER-to-Golgi transport in vitro. A, in vitro ER-to-Golgi transport assay was performed either on ice (lane 1) or at 32 °C (lanes 2-10) in the absence (lane 2) or presence of rat liver cytosol (lanes 1 and 3-10) supplemented with the indicated amounts of GST antibodies (lanes 4-6) or Sec34 antibodies (lanes 7-10). The upper form represents VSVG, whose N-linked glycans are resistant to endoglycosidase H digestion, whereas the lower form represents the ER form, whose N-linked glycans have been removed by endoglycosidase H. B, in vitro transport was performed either on ice (lane 1) or at 32 °C (lanes 2-7) in the absence (lane 2) or presence of rat liver cytosol (lanes 1 and 3-7). The inhibition exhibited by anti-Sec34 (7 µg) was neutralized by a noninhibitory amount of GST-Sec34/F1 (3 µg) (lanes 5 and 7) but not by GST (3 µg) (lanes 4 and 6).

Co-immunoprecipitation of GTC-90, ldlBp, Dor1, and Cod1 from Total Cytosol by Anti-Sec34 Antibodies-- To define the molecular mechanism underlying the action of Sec34, we have performed large-scale immunoprecipitations using rat liver cytosol (Fig. 3). As compared with rabbit anti-goat IgG (Fig. 3, lane 2), antibodies against Sec34 immunoprecipitated several distinct polypeptides (lane 3) as resolved by SDS-PAGE. We focused our mass spectrometric analyses on polypeptides that are larger than the IgG heavy chain because polypeptides of smaller sizes are commonly detected by large-scale immunoprecipitations due to nonspecific interactions of abundant cytosolic proteins. As shown in Fig. 3, GTC-90 (21) and ldlBp (28), in addition to Sec34, were identified in the Sec34 immunoprecipitates. The intensities of ldlBp and GTC-90, as revealed by Coomassie Blue staining, were stronger than that of Sec34, suggesting that the Sec34 polypeptide might be poorly stained or that other subunits are present at higher abundance in the complex. The quantitative and specific recoveries of ldlBp and GTC-90 by anti-Sec34 antibodies from total cytosol suggest that Sec34, GTC-90, and ldlBp are components of the same protein complex(es). Furthermore, two mammalian homologues (Dor1 and Cod1) of the yeast Sec34p-Sec35p complex (32) were also detected in the Sec34 immunoprecipitates (Fig. 3), supporting the hypothesis that they are part of the mammalian complex. To rule out the possibility that some interesting components may be overlooked, we have also analyzed other polypeptides migrating faster than that of IgG heavy chain. These mainly represented abundant cytosolic proteins such as actin, three ribosomal subunits, glyceraldehyde-3-phosphate dehydrogenase, and glutathione peroxidase. In addition, an uncharacterized protein with GenBank^TM accession no. XP_034431 was identified. Although we cannot exclude the possibility that some of these proteins may be involved in Sec34 function, the presence of abundant cytosolic proteins such as actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal proteins in this low molecular weight region indicates that their presence is likely due to nonspecific interactions.

View larger version (64K): [in this window] [in a new window]   Fig. 3.   Co-immunoprecipitation of GTC-90 and ldlBp (as well as Dor1 and Cod1) by anti-Sec34 antibodies. Rat liver cytosol (50 mg) was immunoprecipitated with 500 µg of Sec34 antibodies (lane 3) or rabbit anti-goat IgG (lane 2) immobilized on protein A-Sepharose beads. The immune complexes collected on the beads were washed extensively. The immunoprecipitated proteins were released by boiling in SDS-PAGE sample buffer and then resolved on by 10% SDS-PAGE. Molecular size markers are shown on lane 1. Polypeptides larger than the IgG heavy chain were first excised and subjected to mass spectrometric analysis. The amino acid sequences of tryptic peptides of ldlBp, GTC-90, Dor1, and Cod1 are indicated on the right. The identity of the Sec34 band was established by immunoblotting analysis. Mass spectrometric analysis of polypeptides smaller than the IgG heavy chain was performed and revealed the presence of abundant cytosolic proteins that could be due to nonspecific interactions.

Co-immunoprecipitation of ldlCp and Cod2-- While our study was in progress, the yeast Sec34p-Sec35p complex was characterized in detail (32). It contains eight subunits: Sec34p, Sec35p, Dor1p, Cod1p, Cod2p, Cod3p, Cod4p, and Cod5p. Cod4p corresponds to GTC-90 in mammalian cells. Some structural relatedness between Sec35p and ldlCp was noticed, indicating that Sec35p could be a candidate for the functional counterpart of mammalian ldlCp (32). Mammalian homologues of Dor1p, Cod1p, and Cod2p, but not Cod3p or Cod5p, were also identified, and the epitope-tagged mammalian Dor1 and Cod1 were detected in the Golgi apparatus upon transient expression by transfection (32). No apparent structural or functional yeast counterpart of ldlBp was found in the yeast Sec34p-Sec35p complex. Because GTC-90, ldlBp, Dor1, and Cod1 were recovered in the anti-Sec34 immunoprecipitates, we examined whether other potential subunits (ldlCp and Cod2) of the mammalian complex(es) could be immunoprecipitated by anti-Sec34 antibodies (Fig. 4). 293T cells were transiently transfected with constructs expressing Myc epitope-tagged ldlBp, ldlCp, Dor1, Cod1, or Cod2. Cell lysates were immunoprecipitated with anti-Sec34 antibodies and analyzed by immunoblot to detect the tagged proteins. As positive controls, cells transiently expressing myc-ldlBp, myc-Dor1, and myc-Cod1 were immunoprecipitated with anti-Sec34. About 5% of myc-ldlBp (Fig. 4, lanes 1 and 2), myc-Dor1 (lanes 5 and 6), or myc-Cod1 (lanes 7 and 8) could be recovered by anti-Sec34 antibodies. Under identical conditions, they were not co-immunoprecipitated by other control antibodies (data not shown). These results further support our conclusion that they are specifically co-immunoprecipitated by Sec34 antibodies. Importantly, myc-ldlCp (Fig. 4, lanes 3 and 4) and myc-Cod2 (lanes 9 and 10) were co-immunoprecipitated by anti-Sec34 antibodies with efficiencies comparable to that observed for myc-ldlBp, myc-Dor1, and myc-Cod1, supporting the notion that these other proteins are indeed subunits of the same mammalian complex(es). Consistent with our interpretation that polypeptides smaller than the IgG heavy chain were present in the Sec34 immunoprecipitates due to their high abundance and nonspecific interactions, myc-tagged XP_034431 was not co-immunoprecipiated by anti-Sec34 from lysates prepared from transfected cells (data not shown).

View larger version (77K): [in this window] [in a new window]   Fig. 4.   ldlCp and Cod2 are present in Sec34-containing protein complex. 293T cells were transiently transfected with constructs expressing myc epitope-tagged ldlBp (lanes 1 and 2), ldlCp (lanes 3 and 4), Dor1 (lanes 5 and 6), Cod1 (lanes 7 and 8), or Cod2 (lanes 9 and 10). Cell lysates were immunoprecipitated with Sec34 antibodies. 5% of each lysate (odd-numbered lanes) and the immunoprecipitates (even-numbered lanes) were resolved by SDS-PAGE and analyzed by immnoblot with anti-myc antibodies to detect the co-immunoprecipitated proteins. These myc-tagged proteins were not co-immunoprecipitated by control rabbit IgG or antibodies against Bet3 (data not shown).

Direct Interaction of Sec34 with ldlBp or ldlCp-- We next investigated whether Sec34 could interact directly with any of the other subunits. Myc-Sec34 was co-translated with myc-ldlBp, myc-ldlCp, or myc-Cod2 using the in vitro translation system. The translation reactions were then subjected to immunoprecipitation using Sec34 antibodies or anti-myc antibodies. As shown in Fig. 5, myc-Sec34 and myc-Cod2 were both effectively immunoprecipitated with anti-myc antibodies (lane 3). However, myc-Cod2 was not co-immunoprecipitated by anti-Sec34 under the conditions in which myc-Sec34 was efficiently immunoprecipitated (Fig. 5, lane 2), suggesting that myc-Cod2 and myc-Sec34 do not interact directly. Interestingly, both myc-ldlBp and myc-ldlCp, upon co-translation with myc-Sec34, could be efficiently (30-50% of co-expressed protein) co-immunoprecipitated by anti-Sec34 antibodies (Fig. 5, lanes 5 and 8, respectively), suggesting that both ldlBp and ldlCp could interact directly with Sec34.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Direct interaction of Sec34 with ldlBp and ldlCp. Myc-Sec34 was co-translated with myc-Cod2 (lanes 1-3), myc-ldlBp (lanes 4-6), or myc-ldlCp (lanes 7-9) by in vitro translation reactions in the presence of [35S]methionine. The translation products were immunoprecipitated with either Sec34 antibodies (lanes 2, 5, and 8) or anti-myc antibodies (lanes 3, 6, and 9). The immunoprecipitates and 10% of the respective translation reactions (lanes 1, 4, and 7) were analyzed by SDS-PAGE and fluorography.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sec34, GTC-90, and ldlBp/ldlCp have been independently identified by different approaches. Sec34 was identified based on a genomic approach to search for the mammalian homologue of Sec34p (Ref. 20 and this study), which has a well-defined role in ER-to-Golgi transport in yeast (9-12). GTC-90 was previously identified as a component of a novel protein complex that participates in intra-Golgi transport using an in vitro biochemical assay (21). ldlBp and ldlCp were originally identified by a genetic approach (22-25) and subsequently shown to exist in the same protein complex that regulates Golgi structure and function (28, 30). One of the most important discoveries of the present study is that Sec34, GTC-90, and ldlBp/ldlCp are components of the same protein complex(es) (we tentatively refer to this as the Sec34-GTC-90-ldlBp complex). This is based on several lines of observations. Firstly, GTC-90 and ldlBp (as well as Dor1 and Cod1) could be efficiently and specifically co-immunoprecipitated from rat liver cytosol by antibodies against Sec34. Secondly, upon expression by transient transfection, myc-tagged ldlBp, ldlCp, Dor1, Cod1, and Cod2 could be similarly co-immunoprecipitated by anti-Sec34 antibodies. Finally, direct interaction of Sec34 with ldlBp and ldlCp could be demonstrated in the absence of other subunits using in vitro-translated proteins. While this work was in progress, the yeast Sec34p-Sec35p complex was shown to consist of eight subunits: Sec34p, Sec35p, Dor1p, Cod1p, Cod2p, Cod3p, Cod4p, and Cod5p. Mammalian homologues for Dor1p, Cod1p, Cod2p, and Cod4p, but not Cod3p or Cod5p, have been also identified (32), and GTC-90 appears to be the counterpart of Cod4p. An ~110-amino acid region of ldlCp was found to be homologous to Sec35p, indicating some relatedness between ldlCp and Sec35p (32). Interestingly, no homologue for ldlBp was identified in yeast, suggesting either that there is another subunit that remains to be uncovered in the yeast Sec34p-Sec35p complex or that ldlBp could be a functional counterpart of either Cod3p or Cod5p. Although we favor the possibility that ldlBp (980 amino acids for mouse protein and 962 amino acids for human protein) is not a functional counterpart of Cod3p (417 amino acids) or Cod5p (279 amino acids) due to the lack of similarity of their amino acid sequences and the huge differences in sizes, additional studies are needed to establish this point. Because myc-tagged ldlCp can be co-immunoprecipitated with Sec34 upon expression by transient transfection and a direct interaction between Sec34 and myc-ldlCp could be observed, ldlCp is indeed a component of the Sec34-GTC-90-ldlBp complex. Because ldlCp (731 and 738 amino acids for mouse and human protein, respectively) is much larger than Sec35p (275 amino acids), and the observed sequence homology (24% identity over a region of 110 amino acids) is limited, additional evidence is required to resolve whether ldlCp is the structural and/or functional counterpart of yeast Sec35p. The direct interaction of ldlBp or ldlCp with Sec34 suggests that Sec34 harbors structural information for direct interaction with ldlBp and ldlCp in the absence of other subunits.

The other potential subunits (Dor1, Cod1, and Cod2) of the mammalian Sec34-GTC-90-ldlBp complex were similarly confirmed immunologically and/or biochemically as components of the Sec34-containing protein complex(es). Both Dor1 and Cod1 were recovered in Sec34 immunoprecipitates. Epitope-tagged versions of Dor1, Cod1, and Cod2, upon expression by transient transfection, could be co-immunoprecipitated by anti-Sec34 antibodies at efficiencies comparable to those observed for ldlBp and ldlCp under similar conditions. In summary, six other proteins (ldlBp, ldlCp, Dor1, Cod1, Cod2, and GTC-90/Cod4) in mammalian cells are shown here to be present in the Sec34-containing protein complex(es), with ldlBp and ldlCp having the ability to interact directly with Sec34. It remains possible that these proteins, together with others that have yet to be discovered, may form distinct complexes or subcomplexes. If we assume that a functional counterpart of ldlBp exists in yeast, then at least two other proteins (Cod3 and Cod5) remain to be discovered for the mammalian complex(es). However, if ldlBp is a functional counterpart of Cod3 or Cod5, then we have uncovered all but one of the components of the mammalian complex.

The function of the Sec34-GTC-90-ldlBp complex in mammalian cells remains to be explored. In view of all available results, it could be suggested that the Sec34-GTC-90-ldlBp complex may have a general role in the Golgi apparatus that regulates several trafficking events, including ER-to-Golgi transport, various intra-Golgi transports, and possibly endosome-to-trans-Golgi network traffic. The role of Sec34p and Sec35p in ER-to-Golgi transport has been well established in yeast (9-12). Using a modified assay that reconstitutes synchronized transport of almost all ER-arrested VSVG to the Golgi, it was shown that anti-Sec34 antibodies could exhibit dose-dependent inhibition. Furthermore, the inhibition could be neutralized by a noninhibitory amount of the antigen. We have thus provided evidence that Sec34 plays a similar role in ER-to-Golgi transport in mammalian cells, although the temporal and other mechanistic aspects of its action remain to be explored. Although a role for Sec34p or other subunits in intra-Golgi transport in yeast has yet to be investigated, the purification of GTC-90 complex using an in vitro intra-Golgi transport assay suggests that this complex could be intimately involved in intra-Golgi transport (21). A role for Sec34p in endosome-to-TGN transport has been reported, and this was the basis for which Sec34p was independently identified as Grd20p (41). Although it has been suggested that the role of Sec34p/Grd20p in endosome-to-TGN transport could be due to an indirect effect due to its role in ER-to-Golgi transport (10, 12), the recent identification of the Sec34p complex and the relatedness of this complex with Ypt6p function indicate that the Sec34p-Grd20p complex might have a direct role in endosome-to-TGN transport (32). Detailed studies of this complex and its individual subunits using genetic, biochemical, and cell biochemical approaches in both yeast and mammalian cells will provide additional understanding of its function and mechanism as well as the general organization and regulation of Golgi structure and function.

	ACKNOWLEDGEMENTS

We thank Dr. Sean Munro for the generous gift of constructs expressing myc-tagged Dor1 and Cod1, Kazusa DNA Research Institute for providing KIAA1381 (ldlBp) and KIAA1134 (ldlCp) cDNA clones, the NEDO Human cDNA Sequencing Project for AK026305, and Dr. Tang Bor Luen and Paramjeet Singh for reading the manuscript.

	Note Added in Proof

A recent report has independently demonstrated that Sec34/Cog3, GTC-90/Cog5, ldlBp/Cog1, ldlCp/Cog2, Dor1/Cog8, Cod1/Cog4, Cod2/Cog6, as well as a novel component (Cog7) are components of the same protein complex referred to as conserved oligomeric Golgi (COG) complex with the eight subunits named Cog1-8 (42).

	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.

To whom correspondence should be addressed. Tel.: 65-6778-6827; Fax: 65-6779-1117; E-mail: mcbhwj@imcb.nus.edu.sg.

Published, JBC Papers in Press, April 2, 2002, DOI 10.1074/jbc.M202326200

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TX-100, Triton X-100.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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