Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Oct 1;135(1):53–61. doi: 10.1083/jcb.135.1.53

Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex

PMCID: PMC2121028  PMID: 8858162

Abstract

Coatomer is a cytosolic protein complex that forms the coat of COP I- coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Allen J. B., Walberg M. W., Edwards M. C., Elledge S. J. Finding prospective partners in the library: the two-hybrid system and phage display find a match. Trends Biochem Sci. 1995 Dec;20(12):511–516. doi: 10.1016/s0968-0004(00)89119-7. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Balch W. E., Rothman J. E. Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. Arch Biochem Biophys. 1985 Jul;240(1):413–425. doi: 10.1016/0003-9861(85)90046-3. [DOI] [PubMed] [Google Scholar]
  4. Breeden L., Nasmyth K. Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol. 1985;50:643–650. doi: 10.1101/sqb.1985.050.01.078. [DOI] [PubMed] [Google Scholar]
  5. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cosson P., Démollière C., Hennecke S., Duden R., Letourneur F. Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval. EMBO J. 1996 Apr 15;15(8):1792–1798. [PMC free article] [PubMed] [Google Scholar]
  7. Cosson P., Letourneur F. Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science. 1994 Mar 18;263(5153):1629–1631. doi: 10.1126/science.8128252. [DOI] [PubMed] [Google Scholar]
  8. Duden R., Griffiths G., Frank R., Argos P., Kreis T. E. Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin. Cell. 1991 Feb 8;64(3):649–665. doi: 10.1016/0092-8674(91)90248-w. [DOI] [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. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  11. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  12. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gerich B., Orci L., Tschochner H., Lottspeich F., Ravazzola M., Amherdt M., Wieland F., Harter C. Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3229–3233. doi: 10.1073/pnas.92.8.3229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gietz R. D., Schiestl R. H. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast. 1991 Apr;7(3):253–263. doi: 10.1002/yea.320070307. [DOI] [PubMed] [Google Scholar]
  15. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  16. Hara-Kuge S., Kuge O., Orci L., Amherdt M., Ravazzola M., Wieland F. T., Rothman J. E. En bloc incorporation of coatomer subunits during the assembly of COP-coated vesicles. J Cell Biol. 1994 Mar;124(6):883–892. doi: 10.1083/jcb.124.6.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harrison-Lavoie K. J., Lewis V. A., Hynes G. M., Collison K. S., Nutland E., Willison K. R. A 102 kDa subunit of a Golgi-associated particle has homology to beta subunits of trimeric G proteins. EMBO J. 1993 Jul;12(7):2847–2853. doi: 10.1002/j.1460-2075.1993.tb05946.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harter C., Draken E., Lottspeich F., Wieland F. T. Yeast coatomer contains a subunit homologous to mammalian beta'-COP. FEBS Lett. 1993 Oct 11;332(1-2):71–73. doi: 10.1016/0014-5793(93)80487-f. [DOI] [PubMed] [Google Scholar]
  19. Harter C., Pavel J., Coccia F., Draken E., Wegehingel S., Tschochner H., Wieland F. Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1902–1906. doi: 10.1073/pnas.93.5.1902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hosobuchi M., Kreis T., Schekman R. SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer. Nature. 1992 Dec 10;360(6404):603–605. doi: 10.1038/360603a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kuge O., Hara-Kuge S., Orci L., Ravazzola M., Amherdt M., Tanigawa G., Wieland F. T., Rothman J. E. zeta-COP, a subunit of coatomer, is required for COP-coated vesicle assembly. J Cell Biol. 1993 Dec;123(6 Pt 2):1727–1734. doi: 10.1083/jcb.123.6.1727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Lowe M., Kreis T. E. In vitro assembly and disassembly of coatomer. J Biol Chem. 1995 Dec 29;270(52):31364–31371. doi: 10.1074/jbc.270.52.31364. [DOI] [PubMed] [Google Scholar]
  25. Malhotra V., Serafini T., Orci L., Shepherd J. C., Rothman J. E. Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack. Cell. 1989 Jul 28;58(2):329–336. doi: 10.1016/0092-8674(89)90847-7. [DOI] [PubMed] [Google Scholar]
  26. Murakami Y., Naitou M., Hagiwara H., Shibata T., Ozawa M., Sasanuma S., Sasanuma M., Tsuchiya Y., Soeda E., Yokoyama K. Analysis of the nucleotide sequence of chromosome VI from Saccharomyces cerevisiae. Nat Genet. 1995 Jul;10(3):261–268. doi: 10.1038/ng0795-261. [DOI] [PubMed] [Google Scholar]
  27. Nakayama Y., Goebl M., O'Brine Greco B., Lemmon S., Pingchang Chow E., Kirchhausen T. The medium chains of the mammalian clathrin-associated proteins have a homolog in yeast. Eur J Biochem. 1991 Dec 5;202(2):569–574. doi: 10.1111/j.1432-1033.1991.tb16409.x. [DOI] [PubMed] [Google Scholar]
  28. Neer E. J., Schmidt C. J., Nambudripad R., Smith T. F. The ancient regulatory-protein family of WD-repeat proteins. Nature. 1994 Sep 22;371(6495):297–300. doi: 10.1038/371297a0. [DOI] [PubMed] [Google Scholar]
  29. Nilsson T., Jackson M., Peterson P. A. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell. 1989 Aug 25;58(4):707–718. doi: 10.1016/0092-8674(89)90105-0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  34. Radice P., Pensotti V., Jones C., Perry H., Pierotti M. A., Tunnacliffe A. The human archain gene, ARCN1, has highly conserved homologs in rice and Drosophila. Genomics. 1995 Mar 1;26(1):101–106. doi: 10.1016/0888-7543(95)80087-3. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  37. Serafini T., Orci L., Amherdt M., Brunner M., Kahn R. A., Rothman J. E. ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein. Cell. 1991 Oct 18;67(2):239–253. doi: 10.1016/0092-8674(91)90176-y. [DOI] [PubMed] [Google Scholar]
  38. Serafini T., Stenbeck G., Brecht A., Lottspeich F., Orci L., Rothman J. E., Wieland F. T. A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin. Nature. 1991 Jan 17;349(6306):215–220. doi: 10.1038/349215a0. [DOI] [PubMed] [Google Scholar]
  39. Stenbeck G., Harter C., Brecht A., Herrmann D., Lottspeich F., Orci L., Wieland F. T. beta'-COP, a novel subunit of coatomer. EMBO J. 1993 Jul;12(7):2841–2845. doi: 10.1002/j.1460-2075.1993.tb05945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stenbeck G., Schreiner R., Herrmann D., Auerbach S., Lottspeich F., Rothman J. E., Wieland F. T. Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p. FEBS Lett. 1992 Dec 14;314(2):195–198. doi: 10.1016/0014-5793(92)80973-k. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Waters M. G., Serafini T., Rothman J. E. 'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature. 1991 Jan 17;349(6306):248–251. doi: 10.1038/349248a0. [DOI] [PubMed] [Google Scholar]
  44. Wuestehube L. J., Duden R., Eun A., Hamamoto S., Korn P., Ram R., Schekman R. New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the endoplasmic reticulum to the Golgi complex. Genetics. 1996 Feb;142(2):393–406. doi: 10.1093/genetics/142.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES