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. 1996 Oct;7(10):1535–1546. doi: 10.1091/mbc.7.10.1535

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

J P Paccaud 1, W Reith 1, J L Carpentier 1, M Ravazzola 1, M Amherdt 1, R Schekman 1, L Orci 1
PMCID: PMC276004  PMID: 8898360

Abstract

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Aridor M., Bannykh S. I., Rowe T., Balch W. E. Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport. J Cell Biol. 1995 Nov;131(4):875–893. doi: 10.1083/jcb.131.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  4. Barlowe C., Orci L., Yeung T., Hosobuchi M., Hamamoto S., Salama N., Rexach M. F., Ravazzola M., Amherdt M., Schekman R. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994 Jun 17;77(6):895–907. doi: 10.1016/0092-8674(94)90138-4. [DOI] [PubMed] [Google Scholar]
  5. Barlowe C., Schekman R. SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER. Nature. 1993 Sep 23;365(6444):347–349. doi: 10.1038/365347a0. [DOI] [PubMed] [Google Scholar]
  6. Barlowe C., d'Enfert C., Schekman R. Purification and characterization of SAR1p, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum. J Biol Chem. 1993 Jan 15;268(2):873–879. [PubMed] [Google Scholar]
  7. Bednarek S. Y., Ravazzola M., Hosobuchi M., Amherdt M., Perrelet A., Schekman R., Orci L. COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast. Cell. 1995 Dec 29;83(7):1183–1196. doi: 10.1016/0092-8674(95)90144-2. [DOI] [PubMed] [Google Scholar]
  8. Dascher C., Balch W. E. Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J Biol Chem. 1994 Jan 14;269(2):1437–1448. [PubMed] [Google Scholar]
  9. Duden R., Hosobuchi M., Hamamoto S., Winey M., Byers B., Schekman R. Yeast beta- and beta'-coat proteins (COP). Two coatomer subunits essential for endoplasmic reticulum-to-Golgi protein traffic. J Biol Chem. 1994 Sep 30;269(39):24486–24495. [PubMed] [Google Scholar]
  10. Hicke L., Yoshihisa T., Schekman R. Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum. Mol Biol Cell. 1992 Jun;3(6):667–676. doi: 10.1091/mbc.3.6.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaiser C. A., Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 1990 May 18;61(4):723–733. doi: 10.1016/0092-8674(90)90483-u. [DOI] [PubMed] [Google Scholar]
  12. Kuge O., Dascher C., Orci L., Rowe T., Amherdt M., Plutner H., Ravazzola M., Tanigawa G., Rothman J. E., Balch W. E. Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments. J Cell Biol. 1994 Apr;125(1):51–65. doi: 10.1083/jcb.125.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Letourneur F., Gaynor E. C., Hennecke S., Démollière C., Duden R., Emr S. D., Riezman H., Cosson P. Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell. 1994 Dec 30;79(7):1199–1207. doi: 10.1016/0092-8674(94)90011-6. [DOI] [PubMed] [Google Scholar]
  14. Oprins A., Duden R., Kreis T. E., Geuze H. J., Slot J. W. Beta-COP localizes mainly to the cis-Golgi side in exocrine pancreas. J Cell Biol. 1993 Apr;121(1):49–59. doi: 10.1083/jcb.121.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Orci L., Glick B. S., Rothman J. E. A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Cell. 1986 Jul 18;46(2):171–184. doi: 10.1016/0092-8674(86)90734-8. [DOI] [PubMed] [Google Scholar]
  16. Orci L., Malhotra V., Amherdt M., Serafini T., Rothman J. E. Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack. Cell. 1989 Feb 10;56(3):357–368. doi: 10.1016/0092-8674(89)90239-0. [DOI] [PubMed] [Google Scholar]
  17. Orci L., Palmer D. J., Ravazzola M., Perrelet A., Amherdt M., Rothman J. E. Budding from Golgi membranes requires the coatomer complex of non-clathrin coat proteins. Nature. 1993 Apr 15;362(6421):648–652. doi: 10.1038/362648a0. [DOI] [PubMed] [Google Scholar]
  18. Orci L., Perrelet A., Ravazzola M., Amherdt M., Rothman J. E., Schekman R. Coatomer-rich endoplasmic reticulum. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11924–11928. doi: 10.1073/pnas.91.25.11924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Orci L., Ravazzola M., Meda P., Holcomb C., Moore H. P., Hicke L., Schekman R. Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8611–8615. doi: 10.1073/pnas.88.19.8611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Orcl L., Palmer D. J., Amherdt M., Rothman J. E. Coated vesicle assembly in the Golgi requires only coatomer and ARF proteins from the cytosol. Nature. 1993 Aug 19;364(6439):732–734. doi: 10.1038/364732a0. [DOI] [PubMed] [Google Scholar]
  21. Ostermann J., Orci L., Tani K., Amherdt M., Ravazzola M., Elazar Z., Rothman J. E. Stepwise assembly of functionally active transport vesicles. Cell. 1993 Dec 3;75(5):1015–1025. doi: 10.1016/0092-8674(93)90545-2. [DOI] [PubMed] [Google Scholar]
  22. Pelham H. R. About turn for the COPs? Cell. 1994 Dec 30;79(7):1125–1127. doi: 10.1016/0092-8674(94)90002-7. [DOI] [PubMed] [Google Scholar]
  23. Pepperkok R., Scheel J., Horstmann H., Hauri H. P., Griffiths G., Kreis T. E. Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell. 1993 Jul 16;74(1):71–82. doi: 10.1016/0092-8674(93)90295-2. [DOI] [PubMed] [Google Scholar]
  24. Roth J., Bendayan M., Orci L. Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem. 1978 Dec;26(12):1074–1081. doi: 10.1177/26.12.366014. [DOI] [PubMed] [Google Scholar]
  25. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  26. Rothman J. E., Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. doi: 10.1038/355409a0. [DOI] [PubMed] [Google Scholar]
  27. Salama N. R., Yeung T., Schekman R. W. The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins. EMBO J. 1993 Nov;12(11):4073–4082. doi: 10.1002/j.1460-2075.1993.tb06091.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schekman R., Orci L. Coat proteins and vesicle budding. Science. 1996 Mar 15;271(5255):1526–1533. doi: 10.1126/science.271.5255.1526. [DOI] [PubMed] [Google Scholar]
  29. Shaywitz D. A., Orci L., Ravazzola M., Swaroop A., Kaiser C. A. Human SEC13Rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum. J Cell Biol. 1995 Mar;128(5):769–777. doi: 10.1083/jcb.128.5.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shen K. A., Hammond C. M., Moore H. P. Molecular analysis of SAR1-related cDNAs from a mouse pituitary cell line. FEBS Lett. 1993 Dec 13;335(3):380–385. doi: 10.1016/0014-5793(93)80423-r. [DOI] [PubMed] [Google Scholar]
  31. Steimle V., Siegrist C. A., Mottet A., Lisowska-Grospierre B., Mach B. Regulation of MHC class II expression by interferon-gamma mediated by the transactivator gene CIITA. Science. 1994 Jul 1;265(5168):106–109. doi: 10.1126/science.8016643. [DOI] [PubMed] [Google Scholar]
  32. Tanigawa G., Orci L., Amherdt M., Ravazzola M., Helms J. B., Rothman J. E. Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi-derived COP-coated vesicles. J Cell Biol. 1993 Dec;123(6 Pt 1):1365–1371. doi: 10.1083/jcb.123.6.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tokuyasu K. T. Application of cryoultramicrotomy to immunocytochemistry. J Microsc. 1986 Aug;143(Pt 2):139–149. doi: 10.1111/j.1365-2818.1986.tb02772.x. [DOI] [PubMed] [Google Scholar]
  34. Wadhwa R., Kaul S. C., Komatsu Y., Ikawa Y., Sarai A., Sugimoto Y. Identification and differential expression of yeast SEC23-related gene (Msec23) in mouse tissues. FEBS Lett. 1993 Jan 4;315(2):193–196. doi: 10.1016/0014-5793(93)81161-r. [DOI] [PubMed] [Google Scholar]
  35. Whiteheart S. W., Griff I. C., Brunner M., Clary D. O., Mayer T., Buhrow S. A., Rothman J. E. SNAP family of NSF attachment proteins includes a brain-specific isoform. Nature. 1993 Mar 25;362(6418):353–355. doi: 10.1038/362353a0. [DOI] [PubMed] [Google Scholar]
  36. Yoshihisa T., Barlowe C., Schekman R. Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum. Science. 1993 Mar 5;259(5100):1466–1468. doi: 10.1126/science.8451644. [DOI] [PubMed] [Google Scholar]
  37. Zhang C. J., Rosenwald A. G., Willingham M. C., Skuntz S., Clark J., Kahn R. A. Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo. J Cell Biol. 1994 Feb;124(3):289–300. doi: 10.1083/jcb.124.3.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. d'Enfert C., Gensse M., Gaillardin C. Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast. EMBO J. 1992 Nov;11(11):4205–4211. doi: 10.1002/j.1460-2075.1992.tb05514.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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