In Vivo Assembly of Coatomer, the COP-I Coat Precursor*
- From the Département de Biologie Cellulaire, Université de Genève, Sciences III, 30, quai Ernest-Ansermet, CH-1211 Geneva, Switzerland
- § To whom correspondence should be addressed. Tel.: 41-22-702-6747; Fax: 41-22-781-1747; E-mail: kreis{at}sc2a.unige.ch.
Abstract
Coatomer, a seven-subunit hetero-oligomeric complex, is the major component of the COP-I membrane coat of transport vesicles of the early secretory pathway. We have followed the assembly of this complex in vivo by pulse-chase experiments and immunoprecipitation of native coatomer subunits and found that it is an ordered process that takes 1-2 h to complete. During assembly, direct interactions between α-, β′- and δ-COP, β- and δ-COP, and γ-, ζ-, and δ-COP occur. Coatomer, once it has assembled, is stable with a half-life of ∼28 h. No significant amounts of partial coatomer complexes have been detected. The only subunit to exist at steady state out of the complex is ζ-COP, which has a similar half-life to coatomer subunits within the complex. Assembly is inhibited by brefeldin A, suggesting that it may be a regulated process. These results describe for the first time in vivo assembly of a coat protein complex involved in membrane traffic and extend our knowledge of how coatomer is structured.
Footnotes
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↵‡ Supported by a Travelling Research Fellowship from the Wellcome Trust.
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↵* This work was supported by a grant from the Fonds National Suisse (31-33546.92) (to T. E. K.) and by the Canton de Genève. 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.
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↵1 The abbreviations used are:
- ARF
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ADP-ribosylation factor
- BFA
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brefeldin A
- MEM
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minimum essential medium
- PAGE
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polyacrylamide gel electrophoresis.
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↵2 J. Scheel, R. Pepperkok, M. Lowe, G. Griffiths, and T. E. Kreis, submitted for publication.
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- Received June 14, 1996.
- Revision received July 26, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
