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• W1976177776 abstract "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 A1-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. 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 A1-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. Protein transport between intracellular membrane compartments is mediated by vesicles/tubules that bud from the donor membrane and move to and fuse with the target membrane (reviewed in Bonifacino and Glick (1Bonifacino J.S. Glick B.S. Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1271) Google Scholar) and Lee et al. (2Lee M.C.S. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (695) Google Scholar)). Newly synthesized proteins exit the endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERES, ER exit sites; ERGIC, ER-Golgi intermediate compartment; COPII, coat protein complex II; β-COP, β-coat protein; aa, amino acid; BFA, brefeldin A; Endo H, endoglycosidase H; GFP, green fluorescent protein; GST, glutathione S-transferase; siRNA, short interfering RNA; VSVG, vesicular stomatitis virus-encoded glycoprotein; PA-PLA1, phosphatidic acid-preferring phospholipase A1; MS, mass spectrometry; MALDI-QqTOF, matrix-assisted laser desorption/ionization-quadrupole time of flight. 2The abbreviations used are: ER, endoplasmic reticulum; ERES, ER exit sites; ERGIC, ER-Golgi intermediate compartment; COPII, coat protein complex II; β-COP, β-coat protein; aa, amino acid; BFA, brefeldin A; Endo H, endoglycosidase H; GFP, green fluorescent protein; GST, glutathione S-transferase; siRNA, short interfering RNA; VSVG, vesicular stomatitis virus-encoded glycoprotein; PA-PLA1, phosphatidic acid-preferring phospholipase A1; MS, mass spectrometry; MALDI-QqTOF, matrix-assisted laser desorption/ionization-quadrupole time of flight. in COPII-coated vesicles, which are generated at ER subdomains, known as ER exit sites (ERES), in many eukaryotes (3Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1996; 135: 19-35Crossref PubMed Scopus (330) Google Scholar, 4Rossanese O.W. Soderholm J. Bevis B.J. Sears I.B. O'Connor J. Williamson E.K. Glick B.S. J. Cell Biol. 1999; 145: 69-81Crossref PubMed Scopus (256) Google Scholar). COPII consists of heterotrimeric complexes, Sec23-Sec24, Sec13-Sec31, and a low molecular weight GTP-binding protein Sar1 (5Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar). Sec12, an integral membrane protein of the ER (6Nakano A. Brada D. Schekman R. J. Cell Biol. 1988; 107: 851-863Crossref PubMed Scopus (210) Google Scholar), catalyzes nucleotide exchange on Sar1 (7Barlowe C. Schekman R. Nature. 1993; 365: 347-349Crossref PubMed Scopus (359) Google Scholar, 8Weissman J.T. Plutner H. Balch W.E. Traffic. 2001; 2: 465-475Crossref PubMed Scopus (81) Google Scholar), leading to the exposition of its N-terminal amphipathic domain and membrane binding (9Bielli A. Haney C. Gabreski G. Watkins S.C. Bannykh S.I. Aridor M. J. Cell Biol. 2005; 171: 919-924Crossref PubMed Scopus (166) Google Scholar, 10Lee M.C.S. Orci L. Hamamoto S. Futai E. Ravazzola M. Schekman R. Cell. 2005; 122: 605-617Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). Sar1 activation results in the sequential recruitment of the Sec23-Sec24 complex and the Sec13-Sec31 complex to ER membranes (5Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 11Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) and initiates membrane curvature and constriction to support vesicle fission (9Bielli A. Haney C. Gabreski G. Watkins S.C. Bannykh S.I. Aridor M. J. Cell Biol. 2005; 171: 919-924Crossref PubMed Scopus (166) Google Scholar, 10Lee M.C.S. Orci L. Hamamoto S. Futai E. Ravazzola M. Schekman R. Cell. 2005; 122: 605-617Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The recruited coat subunits Sar1 and Sec23-Sec24 form a prebudding complex on ER membranes. Cargo is sorted into nascent COPII vesicles via interaction with the prebudding complex components Sec24 or its homologues (12Miller E. Antonny B. Hamamoto S. Schekman R. EMBO J. 2002; 21: 6105-6113Crossref PubMed Scopus (196) Google Scholar, 13Mossessova E. Bickford L.C. Goldberg J. Cell. 2003; 114: 483-495Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 14Miller E.A. Beilharz T.H. Malkus P.N. Lee M.C.S. Hamamoto S. Orci L. Schekman R. Cell. 2003; 114: 497-509Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) and/or Sar1 (15Aridor M. Fish K.N. Bannykh S. Weissman J. Roberts T.H. Lippincott-Schwartz J. Balch W.E. J. Cell Biol. 2001; 152: 213-229Crossref PubMed Scopus (203) Google Scholar, 16Giraudo C.G. Maccioni H.J. Mol. Biol. Cell. 2003; 14: 3753-3766Crossref PubMed Scopus (160) Google Scholar, 17Stephens D.J. Pepperkok R. J. Cell Sci. 2004; 117: 3635-3644Crossref PubMed Scopus (32) Google Scholar). The Sec13-Sec31 complex, which can self-assemble to form minimal cages (18Stagg S.M. Gurkan C. Fowler D.M. LaPointe P. Foss T.R. Potter C.S. Carragher B. Balch W.E. Nature. 2006; 439: 234-238Crossref PubMed Scopus (238) Google Scholar), occupies the outermost layer of the coat covering an inner layer of the Sec23-Sec24 complex (19Matsuoka K. Schekman R. Orci L. Heuser J.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13705-13709Crossref PubMed Scopus (94) Google Scholar). Sec23 exhibits GTPase-activating protein activity toward Sar1 (20Yoshihisa T. Barlowe C. Schekman R. Science. 1993; 259: 1466-1468Crossref PubMed Scopus (285) Google Scholar), which results in the depolymerization of the COPII coat, thereby leading to vesicle uncoating that can take place during or after vesicle fission (21Mironov A.A. Mironov Jr., A.A. Beznoussenko G.V. Trucco A. Lupetti P. Smith J.D. Geerts W.J. Koster A.J. Burger K.N. Martone M.E. Deerinck T.J. Ellisman M.H. Luini A. Dev. Cell. 2003; 5: 583-594Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 22Zeuschner D. Geerts W.J. van Donselaar E. Humbel B.M. Slot J.W. Koster A.J. Klumperman J. Nat. Cell Biol. 2006; 8: 377-383Crossref PubMed Scopus (142) Google Scholar).ERES, defined cellular sites that support ER export, have been studied using mammalian cells and yeast Pichia pastoris (23Hammond A.T. Glick B.S. Mol. Biol. Cell. 2000; 11: 3013-3030Crossref PubMed Scopus (210) Google Scholar, 24Bevis B.J. Hammaond A.T. Reinke C.A. Glick B.S. Nat. Cell Biol. 2002; 4: 750-756Crossref PubMed Scopus (186) Google Scholar, 25Stephens D.J. EMBO Rep. 2003; 4: 210-217Crossref PubMed Scopus (84) Google Scholar). ERES can be generated de novo and are relatively immobile structures within the cells. COPII is dynamically exchanged at these sites supporting vesicle budding. Studies by Glick and colleagues (26Soderholm J. Bhattacharyya D. Strongin D. Markovitz V. Connerly P.L. Reinke C.A. Glick B.S. Dev. Cell. 2004; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Connerly P.L. Esaki M. Montegna E.A. Strongin D.E. Levi S. Soderholm J. Glick B.S. Curr. Biol. 2005; 15: 1439-1447Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) have shown that a tightly ER-bound peripheral membrane protein, Sec16, is required for the organization of ERES in P. pastoris.In Saccharomyces cerevisiae, in which well organized ERES are not present, Sec16 potentiates the action of COPII components to bud transport vesicles (28Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (145) Google Scholar, 29Supek F. Madden D.T. Hamamoto S. Orci L. Schekman R. J. Cell Biol. 2002; 158: 1029-1038Crossref PubMed Scopus (102) Google Scholar). A structural difference between P. pastoris and S. cerevisiae Sec16 proteins may explain why higher-order ERES exist in the former yeast but not in the latter one (27Connerly P.L. Esaki M. Montegna E.A. Strongin D.E. Levi S. Soderholm J. Glick B.S. Curr. Biol. 2005; 15: 1439-1447Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).We previously isolated two mammalian Sec23-interacting proteins (p125 and p250) by using glutathione beads coupled to glutathione S-transferase (GST)-mouse Sec23A (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). p125, which appears to be only expressed in mammals, contains an N-terminal Pro-rich region responsible for the interaction with Sec23 (31Mizoguchi T. Nakajima K. Hatsuzawa K. Nagahama M. Hauri H.-P. Tagaya M. Tani K. Biochem. Biophys. Res. Commun. 2000; 279: 144-149Crossref PubMed Scopus (26) Google Scholar), as well as central and C-terminal regions, which exhibit significant sequence homology with phospholipid-modifying proteins, especially phosphatidic acid-preferring phospholipase A1 (PA-PLA1) (32Higgs H.N. Han M.H. Johnson G.E. Glomset J.A. J. Biol. Chem. 1998; 273: 5468-5477Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). p125, PA-PLA1, and KIAA0725p constitute a family of phospholipase A1 proteins that are localized in different cellular compartments and perhaps have different functions (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 31Mizoguchi T. Nakajima K. Hatsuzawa K. Nagahama M. Hauri H.-P. Tagaya M. Tani K. Biochem. Biophys. Res. Commun. 2000; 279: 144-149Crossref PubMed Scopus (26) Google Scholar, 32Higgs H.N. Han M.H. Johnson G.E. Glomset J.A. J. Biol. Chem. 1998; 273: 5468-5477Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 33Nakajima K. Sonoda H. Mizoguchi T. Aoki J. Arai H. Nagahama M. Tagaya M. Tani K. J. Biol. Chem. 2002; 277: 11329-11335Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Although PA-PLA1 and KIAA0725p display phospholipase A1 activity under certain conditions in vitro, p125 does not (32Higgs H.N. Han M.H. Johnson G.E. Glomset J.A. J. Biol. Chem. 1998; 273: 5468-5477Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our recent study demonstrated that p125 is localized at ERES and contributes to their organization (34Shimoi W. Ezawa I. Nakamoto K. Uesaki S. Gabreski G. Aridor M. Yamamoto A. Nagahama M. Tagaya M. Tani K. J. Biol. Chem. 2005; 280: 10141-10148Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).In the present study, we isolated p250 from mouse brain and revealed it to be a protein encoded by the KIAA0310 clone. KIAA0310p, which shows sequence similarity to yeast Sec16 protein, was found to be localized at ERES, and its depletion induced their disorganization. Pull-down experiments demonstrated that KIAA0310p differs from p125 in terms of binding to COPII coat proteins. In addition, overexpression of the two proteins differently affected the organization of ERES.EXPERIMENTAL PROCEDURESAntibodies and Chemicals—To produce an anti-peptide antibody against human KIAA0310p, a mixture of two peptides with an extra cysteine residue at the C-terminal or N-terminal region (MFFQGGETENEENLC and CRLGRIGQRKHLVLN) was conjugated to keyhole limpet hemocyanin and injected into rabbits. The anti-KIAA0310p antibody was affinity-purified from the sera using antigen peptide-coupled beads. Monoclonal (M2) and polyclonal antibodies against FLAG were obtained from Sigma-Aldrich. A polyclonal antibody against GST was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against GM130 and Sec31A were purchased from BD Transduction Laboratories. Polyclonal antibodies against vesicular stomatitis virus-encoded glycoprotein (VSVG), β-COP, Sec23, and Sec31A were prepared in this laboratory (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 34Shimoi W. Ezawa I. Nakamoto K. Uesaki S. Gabreski G. Aridor M. Yamamoto A. Nagahama M. Tagaya M. Tani K. J. Biol. Chem. 2005; 280: 10141-10148Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 35Tani K. Oyama Y. Hatsuzawa K. Tagaya M. FEBS Lett. 1999; 447: 247-250Crossref PubMed Scopus (9) Google Scholar). A monoclonal antibody against ERGIC-53 was kindly provided by Dr. H.-P. Hauri (University of Basel, Switzerland). Brefeldin A (BFA) was obtained from Sigma-Aldrich. Endoglycosidase H (Endo H) was purchased from New England Biolabs (Beverly, MA). Glutathione-Sepharose 4B beads were from Amersham Biosciences (Uppsala, Sweden).Preparation of Mouse Brain Lysates and GST-Sec23A—Mouse brain lysates were prepared as described previously (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). GST-mouse Sec23A was expressed in Sf9 cells and isolated using glutathione beads (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).Peptide Mass Mapping and Data Analysis—The isolated p250 was digested in-gel with trypsin and subjected to peptide mass mapping on a MALDI-QqTOF tandem MS QStar Pulsar I (Applied Biosystems, Foster City, CA). The data were analyzed against the NCBInr protein data base by using the Mascot program.Cell Culture and Subcellular Fractionation—Cell culture was performed as described (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To examine KIAA0310p-depleted HeLa cells by immunofluorescence microscopy, cells were grown on poly-l-lysine-coated coverslips.To determine the subcellular distribution of KIAA0310p, 293T cells were cultured on 10 150-mm dishes, collected, suspended in 3.3 ml of homogenization buffer (10 mm Tris-HCl, pH 7.5, containing 0.25 m sucrose and 1 mm phenylmethylsulfonyl fluoride), and homogenized. The homogenate was centrifuged at 1,000 × g for 10 min, yielding postnuclear supernatant and nuclear fractions. The postnuclear supernatant was then centrifuged at 9,000 × g for 10 min, yielding supernatant and mitochondrial fractions. The supernatant was further centrifuged at 105,000 × g for 1 h, yielding microsomal and cytosolic fractions. Mitochondrial and microsomal pellets were suspended in 300 μl each of homogenization buffer. The nuclear pellet was washed once and suspended in 0.97 ml of buffer. Protein concentrations were determined using the BCA protein assay kit (Pierce Biotechnology).Plasmid Construction, Transfection, and Pull-down Assay—The KIAA0310 clone was obtained from the Kazusa DNA Research Institute (Kazusa, Japan). The obtained clone lacked nucleotides corresponding to the N-terminal 550 amino acids (aa) and corresponding to aa 2,265-2,310. (The full-length cDNA for KIAA0310p, now available from the Kazusa DNA Research Institute, encodes a protein of 2,357 aa.) The C-terminal deletion may be generated by alternative splicing. We constructed a full-length cDNA for KIAA0310p by joining the 5′-end of the cDNA prepared by PCR to the obtained clone. The full-length cDNA for KIAA0310p and a partial cDNA encoding aa 374-2,357 (KIAA0310pΔN), both of which lack aa 2,265-2,310, were inserted into pFLAG-CMV-6c and -6b, respectively, to give rise to the pFLAG-KIAA0310p and pFLAG-KIAA0310pΔN. The mammalian expression plasmids for GST-mouse Sec23A, GST-human Sec24C, FLAG-Sar1(H79G), and FLAG-p125 were constructed previously (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 34Shimoi W. Ezawa I. Nakamoto K. Uesaki S. Gabreski G. Aridor M. Yamamoto A. Nagahama M. Tagaya M. Tani K. J. Biol. Chem. 2005; 280: 10141-10148Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 35Tani K. Oyama Y. Hatsuzawa K. Tagaya M. FEBS Lett. 1999; 447: 247-250Crossref PubMed Scopus (9) Google Scholar). Plasmids for GST-human Sec31A and GST-human Sec13 were prepared in this study using the pEBG vector.For the expression of proteins, HeLa or 293T cells plated on 35-mm dishes were transfected with 1-2 μg of expression plasmids using the Lipofectamine PLUS reagent (Invitrogen) according to the manufacturer's instructions. The pull-down assay was conducted as described (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).Immunofluorescence Microscopy—Immunofluorescence microscopy was performed as described (36Tagaya M. Furuno A. Mizushima S. J. Biol. Chem. 1996; 271: 466-470Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).Short Interfering RNA (siRNA)-mediated Protein Knockdown—The RNA duplexes used for targeting KIAA0310p (oligonucleotide 1, 5′-ACCGCCCAUCGUAAUGAAUUA-3′; oligonucleotide 3, 5′-CCAGGUGUUUAAGUUCAUCUA-3′); and lamin A/C, 5′-AACUGGACUUCCAGAAGAACAUC-3′) were purchased from Japan Bioservice, Inc. Transfection of HeLa cells was performed using Oligofectamine (Invitrogen). The final concentration of an RNA duplex was 200 nm. At 48 h after transfection, the cells were processed for immunoblotting and immunofluorescence.Membrane Binding Assay—Recombinant Sar1 proteins were purified as described previously (37Rowe T. Balch W.E. Methods Enzymol. 1995; 257: 49-53Crossref PubMed Scopus (33) Google Scholar). Microsomal membranes and cytosol were prepared as described previously (38Rowe T. Aridor M. McCaffery J.M. Plutner H. Nuoffer C. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (147) Google Scholar), except that HeLa cells were used instead of rat liver. Membrane binding of KIAA0310p was analyzed by using microsomal membranes (20-40 μg) supplemented with recombinant Sar1 proteins and cytosol, as described for the analysis of Sec23 binding (39Aridor M. Balch W.E. J. Biol. Chem. 2000; 275: 35673-35676Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 40Pathre P. Shome K. Blumental-Perry A. Bielli A. Haney C.J. Alber S. Watkins S.C. Romero G. Aridor M. EMBO J. 2003; 22: 4059-4069Crossref PubMed Scopus (66) Google Scholar).Protein Transport Assay—The expression plasmid for VSVG fused with green fluorescent protein (GFP) was kindly donated by Dr. J. Lippincott-Schwartz (National Institutes of Health, Bethesda, MD). HeLa cells were grown on 35-mm dishes, transfected with duplex RNAs (200 nm), and incubated at 37 °C for 48 h. The cells were then transfected with 1 μg of the plasmid for VSVG-GFP and incubated at 40 °C for 24 h. Cycloheximide was added to a final concentration of 100 μg/ml, and then the cells were shifted to 32 °C to allow transport. At the indicated times, the cells were fixed and processed for immunofluorescence analysis. Alternatively, the cells were solubilized with 0.5% SDS and 1% 2-mercaptoethanol (0.1 ml/35-mm dish) and heated at 100 °C for 10 min. A portion of the lysate was digested with Endo H according to the manufacturer's protocol and then subjected to SDS-PAGE on 8% gels. VSVG was visualized by immunoblotting with a polyclonal anti-VSVG antibody, and the intensities of the immunostained bands were quantitated with NIH image software.RESULTSIdentification of p250—Sec23-interacting proteins were purified from lysates of mouse brain using GST-mouse Sec23A as described previously (30Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). p250 was separated by SDS-PAGE and subjected to MALDI-TOF MS analysis after tryptic digestion. The sequences of more than 10 peptide fragments derived from p250 were found in a protein encoded by the mouse KIAA0310 clone (41Okazaki N. Kikuno R. Ohara R. Inamoto S. Aizawa H. Yuasa S. Nakajima D. Nagase T. Ohara O. Koga H. DNA Res. 2003; 10: 35-48Crossref PubMed Scopus (35) Google Scholar). To confirm that p250 is KIAA0310p, we raised a polyclonal antibody against a mixture of two peptides derived from the sequence of human KIAA0310p and examined whether endogenous KIAA0310p is pulled down with GST-Sec23A expressed in 293T cells. As shown in Fig. 1A, the human p250 pulled down with GST-Sec23A was recognized by the anti-KIAA0310p. The interaction between Sec23A and KIAA0310p was also examined by expressing the two proteins tagged with GST and FLAG, respectively, in 293T cells (Fig. 1B). In this experiment, we used a KIAA0310 construct lacking the N-terminal 373 aa (FLAG-KIAA0310pΔN) because the full-length construct was poorly expressed. As shown in Fig. 1B, FLAG-KIAA0310pΔN was pulled down with GST-Sec23A. The results of these experiments unequivocally indicated that p250 is KIAA0310p.The predicted coding region of KIAA0310 is 2,357 aa long, which is rich in Pro (13.2%), Gly (7.8%), Ser (11.2%), Ala (9.5%), and Leu (8.4%) and includes three Pro-rich regions (aa 3-276, aa 1,113-1,181, and aa 1,931-2,263) and one Tyr-rich region (aa 1,159-1,300). A FASTA search revealed that KIAA0310p shows 19.8% amino acid identity with S. cerevisiae Sec16 protein.KIAA0310p Is Localized at ERES—We next analyzed the localization of KIAA0310p in HeLa cells by immunofluorescence microscopy. As shown in Fig. 2A, immunostaining for KIAA0310p showed a punctuated pattern with some concentration at the perinuclear region, typical for proteins located at ERES. Indeed, KIAA0310p was almost completely colocalized with the COPII component Sec31A (Fig. 2A, top row) and substantially with an ER-Golgi intermediate compartment (ERGIC) marker, ERGIC-53 (middle row). On the other hand, the KIAA0310p-positive puncta, except at the perinuclear region, were negative for the Golgi marker GM130 (bottom row). To confirm the localization of KIAA0310p in ERES, cells were incubated with BFA to disrupt the Golgi apparatus (42Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 16: 1071-1080Crossref Scopus (1537) Google Scholar) or transfected with a GTP-restricted form of Sar1 (Sar1(H79G)) to stabilize and nucleate ERES (43Shima D.T. Cabrera-Poch N. Pepperkok R. Warren G. J. Cell Biol. 1998; 141: 955-966Crossref PubMed Scopus (161) Google Scholar). Although BFA treatment induces Golgi disassembly, peripheral ERES are not significantly affected (34Shimoi W. Ezawa I. Nakamoto K. Uesaki S. Gabreski G. Aridor M. Yamamoto A. Nagahama M. Tagaya M. Tani K. J. Biol. Chem. 2005; 280: 10141-10148Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 44Ward T.H. Polishchuk R.S. Caplan S. Hirschberg K. Lippincott-Schwartz J. J. Cell Biol. 2001; 155: 557-570Crossref PubMed Scopus (345) Google Scholar). As shown in Fig. 2B, BFA treatment induced dispersion of the COPI component β-COP (second row) but did not significantly affect the localization of KIAA0310p (top row) or Sec31A (top and second rows). When the GTP-restricted form of Sar1 was expressed, KIAA0310p (Fig. 2B, third row), like Sec31A (bottom row), accumulated at the perinuclear region, perhaps reflecting the consequence of inhibition of uncoating of the COPII coat.FIGURE 2KIAA0310p is localized at ERES. A, HeLa cells were fixed and double-stained with antibodies against KIAA0310p and Sec31A (top row), ERGIC-53 (middle row), or GM130 (bottom row). Enlarged images of the boxed area are shown. B, HeLa cells were treated with 10 μg/ml BFA for 10 min, fixed, and double-stained with antibodies against Sec31A and KIAA0310p (top row) or β-COP (second row). HeLa cells were transfected with the plasmid for the GTP-restricted form of FLAG-Sar1. At 24 h after transfection, the cells were fixed and double-stained with antibodies against FLAG and KIAA0310p (third row) or Sec31A (bottom row). Merged images are shown on the right. O. E., overexpression. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sar1-dependent Recruitment of KIAA0310p to ER Membranes—In addition to the peripheral and perinuclear dot-like localization, KIAA0310p was shown to be present in the cytosol by immunofluorescence microscopy (data not shown). To confirm this observation, we performed subcellular fractionation. Homogenates of 293T cells were fractionated by differential centrifugation, and each fraction was analyzed by immunoblotting with antibodies against KIAA0310p and Sec23. As shown in Fig. 3A, KIAA0310p was predominantly present in the cytosol. On the other hand, Sec23 was found to be equally distributed in the cytosolic and microsomal fractions. The distribution of KIAA0310p between cytosolic and microsomal fractions was similar when the subcellular fractionation was performed in the absence (Fig. 3A) or presence (not shown) of EDTA.FIGURE 3KIAA0310p is recruited to microsomal membranes in a Sar1-dependent manner. A, subcellular fractionation was performed as described under “Experimental Procedures.” The proteins (25 μg) in each fraction were resolved by SDS-PAGE and then immunoblotted with antibodies against KIAA0310p and Sec23. PNS, postnuclear supernatant (Sup). B, microsomal membranes were incubated with cytosol in the absence (lanes 1 and 5) or presence of 50 ng (lane 2), 250 ng (lane 3), or 1 μg(lane 4) of Sar1(H79G) (Sar1-GTP) or 1 μg(lane 6), 250 ng (lane 7), or 50 ng (lane 8) of Sar1(T39N) (Sar1-GDP). Binding of KIAA0310p and Sec23 to membranes was estimated as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)The finding that KIAA0310p is predominantly cytosolic prompted us to investigate whether the recruitment or association of KIAA0310p to ER membranes is dependent on Sar1, as observed for COPII coat components (5Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 11Matsuoka K. Orci L. Amherdt M. Bednarek S.Y. Hamamoto S. Schekman R. Yeung T. Cell. 1998; 93: 263-275Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 45Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (340) Google Scholar). To this end, m" @default.
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• W1976177776 cites W1499112972 @default.
• W1976177776 cites W1884442241 @default.
• W1976177776 cites W1963573377 @default.
• W1976177776 cites W1971476311 @default.
• W1976177776 cites W1987517959 @default.
• W1976177776 cites W1990364715 @default.
• W1976177776 cites W2003123776 @default.
• W1976177776 cites W2003519864 @default.
• W1976177776 cites W2003993233 @default.
• W1976177776 cites W2005733184 @default.
• W1976177776 cites W2008149441 @default.
• W1976177776 cites W2008395242 @default.
• W1976177776 cites W2012275595 @default.
• W1976177776 cites W2016451610 @default.
• W1976177776 cites W2020212296 @default.
• W1976177776 cites W2022725170 @default.
• W1976177776 cites W2029152767 @default.
• W1976177776 cites W2029983725 @default.
• W1976177776 cites W2030749742 @default.
• W1976177776 cites W2051288907 @default.
• W1976177776 cites W2056717881 @default.
• W1976177776 cites W2059158034 @default.
• W1976177776 cites W2059428912 @default.
• W1976177776 cites W2063672920 @default.
• W1976177776 cites W2067220774 @default.
• W1976177776 cites W2074897257 @default.
• W1976177776 cites W2077553732 @default.
• W1976177776 cites W2078341212 @default.
• W1976177776 cites W2081064935 @default.
• W1976177776 cites W2086084527 @default.
• W1976177776 cites W2089515187 @default.
• W1976177776 cites W2092356580 @default.
• W1976177776 cites W2093546518 @default.
• W1976177776 cites W2099347019 @default.
• W1976177776 cites W2104152706 @default.
• W1976177776 cites W2104360518 @default.
• W1976177776 cites W2105955533 @default.
• W1976177776 cites W2109975639 @default.
• W1976177776 cites W2113439078 @default.
• W1976177776 cites W2116944145 @default.
• W1976177776 cites W2129177835 @default.
• W1976177776 cites W2129913763 @default.
• W1976177776 cites W2130965044 @default.
• W1976177776 cites W2134245600 @default.
• W1976177776 cites W2134576488 @default.
• W1976177776 cites W2140167060 @default.
• W1976177776 cites W2144796443 @default.
• W1976177776 cites W2144918914 @default.
• W1976177776 cites W2160513335 @default.
• W1976177776 cites W2165885405 @default.
• W1976177776 cites W2167837958 @default.
• W1976177776 cites W2169092187 @default.
• W1976177776 cites W2171227030 @default.
• W1976177776 cites W2172109868 @default.
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