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• W1964906411 abstract "COPII proteins are necessary to generate secretory vesicles at the endoplasmic reticulum. In yeast, the Sec24p protein is the only COPII component in which two close orthologues have been identified. By using gene knock-out in yeast, we found that the absence of one of these Sec24 orthologues resulted in a selective secretion defect for a subset of proteins released into the medium. Data base searches revealed the existence of an entire family of Sec24-related proteins in humans, worms, flies, and plants. We identified and cloned two new human cDNAs encoding proteins homologous to yeast Sec24p, in addition to two human cDNAs already present within the data bases. The entire Sec24 family identified to date is characterized by clusters of highly conserved residues within the 2/3 carboxyl-terminal domain of all the proteins and a divergent amino terminus domain. Human (h) Sec24 orthologues co-immunoprecipitate with hSec23Ap and migrate as a complex by size exclusion chromatography. Immunofluorescence microscopy confirmed that these proteins co-localize with hSec23p and hSec13p. Together, our data suggest that in addition to its role in the shaping up of the vesicle, the Sec23-24p complex may be implicated in cargo selection and concentration. COPII proteins are necessary to generate secretory vesicles at the endoplasmic reticulum. In yeast, the Sec24p protein is the only COPII component in which two close orthologues have been identified. By using gene knock-out in yeast, we found that the absence of one of these Sec24 orthologues resulted in a selective secretion defect for a subset of proteins released into the medium. Data base searches revealed the existence of an entire family of Sec24-related proteins in humans, worms, flies, and plants. We identified and cloned two new human cDNAs encoding proteins homologous to yeast Sec24p, in addition to two human cDNAs already present within the data bases. The entire Sec24 family identified to date is characterized by clusters of highly conserved residues within the 2/3 carboxyl-terminal domain of all the proteins and a divergent amino terminus domain. Human (h) Sec24 orthologues co-immunoprecipitate with hSec23Ap and migrate as a complex by size exclusion chromatography. Immunofluorescence microscopy confirmed that these proteins co-localize with hSec23p and hSec13p. Together, our data suggest that in addition to its role in the shaping up of the vesicle, the Sec23-24p complex may be implicated in cargo selection and concentration. endoplasmic reticulum open reading frame amino acids fluorescein isothiocyanate base pair polymerase chain reaction room temperature phosphate-buffered saline human The intracellular transport of secretory proteins from the endoplasmic reticulum (ER)1to the cell surface is essentially mediated by vesicles that collect and concentrate cargo from a donor compartment and deliver it to the subsequent compartment. Each step of vesicle budding, targeting, and fusion implies that resident proteins from the donor compartment must be excluded from the forming vesicle, whereas cargo is selected and concentrated into it (1Rothman J.E. Orci L. Nature. 1992; 355: 409-415Crossref PubMed Scopus (739) Google Scholar, 2Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (809) Google Scholar, 3Schekman R. Barlowe C. Bednarek S. Campbell J. Doering T. Duden R. Kuehn M. Rexach M. Yeung T. Orci L. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 11-21Crossref PubMed Google Scholar). The isolation of several yeast mutants impaired in ER to Golgi transport led to the purification of a set of five cytosolic proteins sufficient to reconstitute the process of ER vesicle formation in vitro (4Kaiser C.A. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (536) Google Scholar, 5Schekman R. Curr. Opin. Cell Biol. 1992; 4: 587-592Crossref PubMed Scopus (114) Google Scholar, 6Barlowe 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 (1025) Google Scholar). This COPII coat complex consists of Sar1p, Sec23-Sec24p, and Sec13-Sec31p complexes (7Pryer N.K. Wuestehube L.J. Schekman R. Annu. Rev. Biochem. 1992; 61: 471-516Crossref PubMed Scopus (368) Google Scholar, 8Salama N.R. Yeung T. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar). Proteins of the secretory machinery appear to be well conserved throughout evolution, as a set of COPII proteins with significant homologies with the yeast proteins was identified in mammalian cells. Two mammalian homologues of yeast Sar1p were identified and found to localize to the transitional zone of the ER (9Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (252) Google Scholar). The human homologue of Sec13p was shown to participate in the exit of VSV-G protein from the ER (10Tang B. Peter F. Krijnse-Locker J. Low S. Griffiths G. Hong W. Mol. Cell. Biol. 1997; 17: 256-266Crossref PubMed Scopus (100) Google Scholar). We recently cloned and characterized two isoforms of human Sec23p, one of which complements a temperature-sensitivesec23-1 mutation (11Paccaud J. Reith W. Carpentier J. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: 1535-1546Crossref PubMed Scopus (96) Google Scholar). The in vitroreconstitution of mammalian ER vesicle formation was documented recently and found to be quite analogous to that in yeast (12Aridor M. Weissman J. Bannykh S. Nuoffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar). During vesicle formation, cargo proteins are concentrated into the budding vesicle, and ER resident proteins are selectively excluded (13Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (331) Google Scholar,14Mizuno M. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5732-5736Crossref PubMed Scopus (114) Google Scholar). Two models can account for this process of cargo selection and concentration into the nascent vesicle. The first model proposes the existence of molecular sieves that allow only secretory molecules to selectively enter the vesicle. The yeast protein Shr3 could be such a sieve for a particular set of yeast proteins, the amino acid permeases (15Kuehn M.J. Schekman R. Ljungdahl P.O. J. Cell Biol. 1996; 135: 585-595Crossref PubMed Scopus (93) Google Scholar). The second model postulates that vesicle coat components interact with putative cargo receptors to sort and concentrate cargo molecules. By interacting either directly with determinants found on secretory membrane proteins or indirectly with putative “cargo receptors,” the COPII coat would thus participate in both cargo selection and bud formation (16Kuehn M.J. Schekman R. Curr. Opin. Cell Biol. 1997; 9: 477-483Crossref PubMed Scopus (105) Google Scholar). The latter model recently received support from work carried out in yeast where it was shown that purified COPII components interact with vesicle integral membrane proteins and soluble cargo (17Kuehn M.J. Herrmann J.M. Schekman R. Nature. 1998; 391: 187-190Crossref PubMed Scopus (321) Google Scholar). Moreover, in mammalian cells the transmembrane VSV-G protein was selectively recruited into the forming vesicle by the pre-budding complex composed of Sar1p and Sec23-24p complex, but no direct interaction between the cytosolic portion of VSV-G with the Sec23-24p complex could be demonstrated (12Aridor M. Weissman J. Bannykh S. Nuoffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (241) Google Scholar). Potential cargo receptors have been tentatively identified recently. One is the p24 family of transmembrane proteins found both in yeast and mammals. These proteins, located essentially between the ER and the Golgi apparatus, recycle between these organelles and are incorporated into both COPI and COPII vesicles (18Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 19Stamnes M. Craighead M. Hoe M. Lampen N. Geromanos S. Tempst P. Rothman J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8011-8015Crossref PubMed Scopus (195) Google Scholar, 20Fiedler K. Rothman J. J. Biol. Chem. 1997; 272: 24739-24742Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 21Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J. Wieland F. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (180) Google Scholar, 22Dominguez M. Dejgaard K. Füllerkrug J. Dahan S. Fazel A. Paccaud J.-P. Thomas D.Y. Bergeron J.J.M. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (287) Google Scholar). However, their role is still unclear, as p23 in particular appeared to be restricted to a structural role rather than in sorting (23Rojo M. Pepperkok R. Emery G. Kellner R. Stang E. Parton R.G. Gruenberg J. J. Cell Biol. 1997; 139: 1119-1135Crossref PubMed Scopus (122) Google Scholar). In yeast, however, the deletion of members of this family delays the secretion of a subset of secretory protein (18Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 24Schimmöller F. Singer-Krüger B. Schröder S. Krüger U. Barlowe C. Riezmann H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (281) Google Scholar, 25Elrod-Erickson M. Kaiser C. Mol. Biol. Cell. 1996; 7: 1043-1058Crossref PubMed Scopus (142) Google Scholar). The cytoplasmic tail of mammalian p24 proteins interacts with coatomer (COPI) (26Fiedler K. Veit M. Stamnes M. Rothman J. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (272) Google Scholar), but we showed recently that it also binds specifically to the mammalian COPII component Sec23Ap via a di-aromatic motif (22Dominguez M. Dejgaard K. Füllerkrug J. Dahan S. Fazel A. Paccaud J.-P. Thomas D.Y. Bergeron J.J.M. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (287) Google Scholar). Another potential candidate as cargo receptor is the mannose-specific lectin-like transmembrane molecule ERGIC53/58. This protein cycles between the ER and the Golgi and is implicated in the forward transport of glycosylated proteins out of the ER (27Itin C. Foguet M. Kappeler F. Klumperman J. Hauri H. Biochem. Soc. Trans. 1995; 23: 541-544Crossref PubMed Scopus (16) Google Scholar, 28Vollenweider F. Kappeler F. Itin C. Hauri H.P. J. Cell Biol. 1998; 142: 377-389Crossref PubMed Scopus (133) Google Scholar). Its short cytosolic tail contains a KKFF retrieval motif which mediates its interaction with the COPI complex (29Schindler R. Itin C. Zerial M. Lottspeich F. Hauri H.P. Eur. J. Cell Biol. 1993; 61: 1-9PubMed Google Scholar), but also with the mammalian Sec23-24p complex, via its di-phenylalanine motif (30Kappeler F. Klopfenstein D.R.C. Foguet M. Paccaud J.-P. Hauri H.-P. J. Biol. Chem. 1997; 272: 31801-31808Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). 2A. Pagano, F. Letourneur, D. Garcia-Estefania, J.-L. Carpentier, L. Orci, and J.-P. Paccaud, unpublished results. 2A. Pagano, F. Letourneur, D. Garcia-Estefania, J.-L. Carpentier, L. Orci, and J.-P. Paccaud, unpublished results. Collectively, these data suggest that COPII components may have a dual role during vesicle biogenesis; they participate both in the deformation of the lipid bilayer to shape up the vesicle and in the sorting of cargo. In this perspective, a likely component to function as an adaptor during the sorting process is the Sec23-24p complex. Interestingly, in the yeast genome at least two additional genes highly related to the essential SEC24 gene can be identified. In the present report, we investigated the role of these yeast genes in secretion, and we identify four new human proteins related to yeast Sec24p. The biochemical and morphological characterization of these proteins enabled us to demonstrate that they are indeed mammalian forms of Sec24p, with their corresponding conserved relatives inCaenorhabditis elegans, Arabidopsis thaliana, andDrosophila melanogaster. Based on our findings, we propose that the multiple Sec23-24p complexes function as adaptors for subclasses of secretory cargo. Biochemicals were purchased from Merck unless stated otherwise; T3 RNA polymerase, rNTPs, and Pfu DNA polymerase were from Promega; restriction enzymes, ligase, and calf intestinal phosphatase were from New England Biolabs; pYES2 and pRSETc vectors were obtained from Invitrogen; pBlue-Script from Stratagene and pCiNeo were obtained from Promega; T7 DNA sequencing kit and Superdex-200 were purchased from Amersham Pharmacia Biotech; pGEX-KG was kindly provided by K. Guan (31Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1635) Google Scholar); anti-rabbit Ig-horseradish peroxidase, anti-mouse Ig-horseradish peroxidase, ECL reagents kit, [32P]UTP, and35S-ATP were from Amersham Pharmacia Biotech; rhodamine-conjugated goat anti-rabbit Ig and FITC-conjugated goat anti-mouse Ig were from Sigma; monoclonal anti-hemagglutinin 12CA5 was from Babco; monoclonal anti-FLAG was purchased from Eastman Kodak Co.; anti-hSec13p antibodies were a kind gift from W. Hong (Singapore). Mammalian cells were cultured in their appropriate medium supplemented with 10% fetal calf serum at 5% CO2. Yeast cultures and manipulations were done according to Guthrie and Fink (32Guthrie C. Fink G.R. Methods Enzymol. 1991; : 194Google Scholar), and unless otherwise stated, molecular biology procedures were performed as described by Ausubel et al. (33Ausubel F.M. Brent R. Kinston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Chanda V.B. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1992Google Scholar). Yeast strains were Saccharomyces cerevisiae RSY607 (MAT alfa, PEP4::URA3 leu2-3, 112 ura3-52) and PJ69-4A (Mat a, trp1-901 leu2-3, 112 ura3-52 his3-200 gal4_ gal4_80 LYS::GAL1-HIS3GAL2-ADE2 met2::GAL7-lacZ;E. coli strains were DH5α and BL21/DE3-LysS. Based on the yeast Sec24 cDNA sequence used to screen ESTs data bases (34Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar, 35Altschul S.F. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5509-5513Crossref PubMed Scopus (389) Google Scholar), two different probes were designed. The oligonucleotide 5′-TTAGCCTTGGACTGTTCTGGTCAGC-3′ derived from EST T79533 was used to screen for hSec24A; the primers 5′-TATTCTGCAGGGTGCATC-3′ and 3′-GAACGGACAAAGAAGTTAC-5′ (positions 221 and 407, respectively) were constructed based on the ESTZ43853 to obtain a 189-bp PCR product. These two probes were used to screen a size-selected human B lymphocyte cDNA library as described previously (11Paccaud J. Reith W. Carpentier J. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: 1535-1546Crossref PubMed Scopus (96) Google Scholar). Sequencing of the cDNA sequences selected was performed by primer walking in both directions. KIAA0079 cDNA was kindly provided to us by Dr. Nomura. The FLAG epitope was introduced at the 5′ end of hSec24B cDNA by PCR from position 159 to position 1349 in the cDNA sequence. The PCR product was subsequently subcloned into the XhoI andNheI sites of the pBSKS-hSec24B to obtain Flag-hSec24B. This epitope-tagged cDNA was subcloned into the mammalian expression vector pCiNeo. A similar strategy was used to produce Flag-hSec24C. All the constructs were verified by sequencing. Gene deletions were carried out according to the method of Baudinet al. (36Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1107) Google Scholar), by PCR amplification of the HIS3gene with oligonucleotides that encode 40 bp of the gene-specific sequences from each end of the open reading frames to be deleted. The 5′-rapid amplification of cDNA ends experiment was performed using the 5′-rapid amplification of cDNA ends kit from Life Technologies, Inc. The antisense gene-specific primer used was 5′-CTGGTTGAAAAGTTGTAGG-3′ (positions 364–382 of hSec24A cDNA). Antibodies against hSec24B were prepared against a glutathione S-transferase fusion protein containing aa 71–445 of hSec24B. The GST-hSec24B fusion construct was obtained by PCR from position 212 to position 1349. This PCR product was cloned into the bacterial expression vector pGEX-KG. Similarly, anti-Sec24C antibodies were obtained against a glutathioneS-transferase fusion protein containing aa 207–494 of the hSec24C. The constructions were verified by sequencing the entire PCR product. Affinity purification of antibodies was performed by coupling the immunogen to CNBr-activated Sepharose according to the manufacturer's indications. The affinity matrix was incubated with crude antiserum for 2 h at 4 °C, washed extensively with 20 mm Tris, pH 6.8, NaCl 150 mm; bound antibodies were eluted with 100 mm glycine HCl, pH 2.0, and fractions were immediately neutralized by the addition of 1 m Tris, pH 8.0. The antibody preparation was dialyzed against PBS, 10% glycerol, 1 mm sodium azide. Cell extracts were either prepared as total Triton X-100 lysate or cytosol. For Triton X-100 extract, 5 × 106 cells were lysed in 1 ml of lysis buffer (20 mm HEPES, pH 6.8, 125 mm potassium acetate, 5 mm MgCl2, 1 mm EDTA, and the following protease inhibitor, Complete™ protease inhibitor mixture tablets, from Boehringer Mannheim). To obtain cytosol, we resuspended HepG2 cells in lysis buffer (20 mm HEPES, pH 6.8, 100 mm potassium acetate, 5 mm MgCl2, 8% sucrose) and homogenized cells using 20 strokes of a tight-fitting glass Potter homogenizer in the presence of 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and protease inhibitors. Postnuclear supernatant was prepared by centrifuging at 2,000 × gfor 10 min at 4 °C. This postnuclear supernatant was then centrifuged at 100,000 × g for 60 min at 4 °C, snap-frozen in liquid nitrogen, and stored at −80 °C. Aliquots of cytosol were fractionated onto a Superdex 200 column, and fractions were analyzed as described previously (45.Deleted in proof.Google Scholar). Immunoprecipitation was performed at 4 °C on 400 μg of Triton X-100 cell extracts using 5 μl of antiserum in a total volume of 500 μl. The precipitate was collected by adding 25 μl of protein A-agarose beads (1:1 slurry), washed five times with lysis buffer and once in 20 mm Tris, pH 6.8. The beads were resuspended in SDS sample buffer and then run on 9% polyacrylamide gels before being transferred to nitrocellulose. Total protein secretion assay was performed as described previously (37Gaynor E.C. Emr S.D. J. Cell Biol. 1997; 136: 789-802Crossref PubMed Scopus (164) Google Scholar). Media proteins were precipitated with ConA-Sepharose 4B (Sigma), and proteins were separated on 8% SDS-polyacrylamide gel electrophoresis. Total RNA from cultured cells was purified using the guanidinium acid-phenol method of Chomczynski and Sacchi (38Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). Probes for hSec24A, -B, and -C were synthesized with T3 RNA polymerase in the presence of 50 μCi of [32P]UTP and purified on acrylamide-urea gel. Probes had a length of 365 (position 293–658 of cDNA), 126 (position 2128–2254), and 282 bp for hSec24A, -B, and -C, respectively. The hybridization, digestion, and analysis of the protected fragments were done as described previously (11Paccaud J. Reith W. Carpentier J. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: 1535-1546Crossref PubMed Scopus (96) Google Scholar). Monolayers of cells were fixed in 4% paraformaldehyde in PBS for 20 min at RT. Cells were either permeabilized by dehydration and rehydration in ethanol or 0.2% saponin in PBS for 15 min. Primary antibodies were incubated for 2 h in a moist chamber at RT, followed by washing with PBS. Secondary antibodies FITC or rhodamine-conjugated goat anti-rabbit or goat anti-mouse IgG were added for 1 h at RT. When appropriate, specific antibodies were first adsorbed with 50 μg of the recombinant immunogen for 30 min at RT prior to incubation with cells. The interaction between the central region of hSec24Cp and hSec23Ap was tested by the yeast two-hybrid method, as described by James et al. (39James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Different portions of hSec24C cDNA were cloned into the vector pGBDU-C2 in frame with the GAL4 binding domain. The constructs prepared for the assay were the following: hSec24C1 (aa 198–807), hSec24C2 (aa 485–807), hSec24C3 (aa 198–694), hSec24C4 (aa 485–694), and hSec24C5 (aa 485–640). The entire coding sequence of hSec23A and two truncated forms, hSec23A/1 (aa 1–543) and hSec23A/2 (aa 1–256), were cloned into the vector pGAD-C1 in frame with the GAL4 activation domain. A search through the yeast genome using the sequence of yeast Sec24 (kindly provided to us by Randy Schekman) revealed the existence of two additional hypothetical proteins related to yeast Sec24p (referred to as YNE09 and YHP8, SWISS-PROT codes p53953 and p38810, respectively). YNE09 and YHP8 share 56 and 23%, respectively, of similarity with the essential yeast geneSEC24. The existence of yeast homologues of Sec24 proteins prompted us to investigate the consequences of their loss on secretion processes. We knocked out YNE09 and YHP8 alone or in combination, and the secretion of known secretory proteins such as invertase and CPY was assessed. We also monitored total protein secretion in the culture medium by pulse-chase experiments. Although the knock-out of yeast Sec24p is lethal (40Gimeno R. Espenshade P. Kaiser C. Mol. Biol. Cell. 1996; 7: 1815-1823Crossref PubMed Scopus (97) Google Scholar), 3T. Yoshihisa and R. Schekman, personal communication. the deletion of the two other orthologues of Sec24p YNE09 and YHP8 were viable, as well as the double knock-out. The pattern of secretion of CPY and invertase of knock-outs were indistinguishable from that of wild-type cells (not shown). We next analyzed the general profile of protein secreted in the supernatant after a pulse-chase with radioactive methionine. In order to potentiate any secretory defect caused by the absence of a given Sec24p, the cells were incubated at 37 °C; the viability of cells with a single as well as a double deletion was not affected by high temperature. However, when we examined the secretory pattern of such cells, selective secretory defects became apparent; the deletion of YHP8 prevented almost entirely the secretion of a small subset of proteins when incubated at 37 °C, a defect already noticed at 30 °C (Fig.1). The major proteins disappearing from the supernatant had a apparent molecular mass of about 55 and 100 kDa, whereas smaller species around 30 kDa were also affected but to a lesser extent (Fig. 1). The identity of the proteins selectively retained is currently under investigation. When we extended our data base search, it became apparent that highly related genes existed in other organisms such as mammals, worms, flies, or plants. We first identified three different human ESTs sharing a significant degree of homology with the probe. One EST pointed to an already cloned cDNA named KIAA0079 (GenBankTM accession number 1723050), which was kindly provided to us by Dr. Nomura. This cDNA encodes a protein of 1125 amino acids with a predicted molecular mass of 121 kDa. We used the remaining two EST sequences to probe a human cDNA library and isolated two human cDNAs: whereas one cDNA contained an apparently complete ORF, the other appeared incomplete at its 5′ end, because it lacked the initiator ATG codon. We performed a rapid amplification of cDNA ends experiment to obtain the 5′ end of the cDNA, and we extended our initial sequence by 360 base pairs, increasing the previous predicted ORF to a putative protein of about 118 kDa and a length of 1078 amino acids. However, the initiator ATG is still missing, and we are attempting alternative methods to obtain the complete ORF. This putative protein was named hSec24Ap (GenBankTM accession number AJ131244). The other cDNA encoded a protein of 1268 amino acids with a predicted molecular mass of 137 kDa and was named hSec24Bp (GenBankTM accession number AJ131245). Recently, an additional mRNA sequence named KIAA0755 (accession number 3882231) was added to GenBankTMand encodes a protein of 1032 amino acids related to our hSec24 sequences. We aligned these four human proteins using the multiple alignment analysis software Clustal W (41Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54908) Google Scholar). We found a strong homology over the carboxyl-terminal two-thirds of their length (Fig.2), and although their amino termini are divergent, the overall similarity between the four proteins is approximately 35%, but it increases up to 45% in the carboxyl-terminal region. From the alignment, hSec24A and -B are more closely related to each other than they are to KIAA0079 and KIAA0755 and vice versa. However, due to the evident similarity of these sequences with the other two human putative Sec24 proteins, we propose to rename KIAA0079 and KIAA0755, respectively, hSec24Cp and hSec24Dp. Sec24-related proteins are found in other organisms as well. InC. elegans, two hypothetical proteins of similar length were identified (GenBankTM accession numbers 1163046 and 137014) that share more than 45% similarity with hSec24Ap and hSec24Cp, respectively. An A. thaliana cDNA (GenBankTM accession number 3063706) encoding for a protein sharing up to 40% similarity with hSec24Cp was found. Finally, we identified a genomic DNA sequence (GenBankTM accession number AC004340) ofD. melanogaster in which putative exons encode a protein sharing more than 65% similarity over ¾ of the length of hSec24Cp. Prosite pattern searching (42Appel R.D. Bairoch A. Hochstrasser D.F. Trends Biochem. Sci. 1994; 19: 258-260Abstract Full Text PDF PubMed Scopus (511) Google Scholar) did not retrieve any significant known protein motif within any member of the family. However, the alignment of all identified members of the Sec24 family revealed interesting features as follows: several entirely conserved positions are found throughout the carboxyl terminal two-thirds of the proteins, and in particular, a highly conserved region consisting of two tandems of cysteines separated by 17 or 18 amino acids reminiscent of a zinc finger-like domain can be considered as the typical signature of the family (underlined in Fig. 2). From the above analysis, we hypothesized that our cloned proteins are human Sec24 homologues. If this assumption is correct, the proteins should be confined to intracellular locations compatible with their putative role in ER vesicle formation, namely the transitional elements of the ER and the intermediate compartment, where other COPII components have been previously localized (10Tang B. Peter F. Krijnse-Locker J. Low S. Griffiths G. Hong W. Mol. Cell. Biol. 1997; 17: 256-266Crossref PubMed Scopus (100) Google Scholar, 11Paccaud J. Reith W. Carpentier J. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: 1535-1546Crossref PubMed Scopus (96) Google Scholar, 43Orci L. Ravazzola M. Meda P. Holcomb C. Moore H.P. Hicke L. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8611-8615Crossref PubMed Scopus (145) Google Scholar, 44Shaywitz D.A. Orci L. Ravazzola M. Swaroop A. Kaiser C.A. J. Cell Biol. 1995; 128: 769-777Crossref PubMed Scopus (85) Google Scholar). In order to verify this hypothesis, we tagged hSec24Bp and hSec24Cp at their amino termini with the FLAG epitope, and we expressed the constructs transiently in different human cell lines. We also isolated stable transfected clones of Chinese hamster ovary cells expressing hSec24Cp. We first verified biochemically that the tagged proteins would behave as expected. Total cell extracts were analyzed by Western blotting with the FLAG monoclonal antibody. For hSec24Bp, a single band migrating at an apparent molecular mass of 140 kDa was detected using anti-FLAG antibodies (Fig. 3 A). The same cell extracts were probed with polyclonal anti-hSec24Bp antibodies generated against a glutathione S-transferase fusion of the amino-terminal portion of hSec24B. This antibody specifically recognized two bands as follows: the slower migrating form having almost the same apparent molecular mass as the epitope-tagged hSec24Bp, and the lower band was estimated to have a molecular mass of about 135 kDa (Fig. 3 A). Affinity purified antibodies still recognized both bands, whereas the detection of both bands was abolished when using immuno-depleted antiserum. This doublet is seen in most human cell lines tested, although the relative intensity of the each band in the doublet appears to be cell type-specific (Fig. 3 B). We don't yet know the meaning of this doublet, but it may likely represent degradation products of the protein. More importantly, this antibody discriminates hSec24Bp from hSec24Cp (Fig. 3 A). The epitope-tagged hSec24Cp migrated at approximately the same molecular mass of about 120 kDa (Fig. 3 A). We then looked at the intracellular distribution of hSec24Bp by immunofluorescence in various human cell lines. By using antibodies against native hSec24Bp, the protein displayed a punctate pattern scattered throughout the cytoplasm along with a labeling around the perinuclear region (Fig. 4 A). The same analysis was done with an epitope-tagged construct transiently expressed in Hela cells; the epitope-tagged hSec24Bp distribution was" @default.
• W1964906411 created "2016-06-24" @default.
• W1964906411 creator A5003320334 @default.
• W1964906411 creator A5017155532 @default.
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• W1964906411 date "1999-03-01" @default.
• W1964906411 modified "2023-10-14" @default.
• W1964906411 title "Sec24 Proteins and Sorting at the Endoplasmic Reticulum" @default.
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