In Vitro Assembly and Disassembly of Coatomer (*)
- 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; Kreis{at}sc2a.unige.ch.
Abstract
Coatomer, a complex of seven proteins, is the major component of the non-clathrin (COP I) membrane coat. We report here the
first system to reversibly disassemble and reassemble this complex in vitro. Coatomer disassembles at high salt concentrations and reassembles when returned to a more physiological buffer. Using this
system, we show that α-, β′-, and 
-COP interact directly and that 
-COP interacts with 
-COP. A partial complex comprising α-, β′-, and 
-COP, obtained after coatomer disassembly, can bind to membranes in vitro. This binding is, at least in part, mediated by interactions with cytoplasmic KKXX motifs of proteins normally retained in or retrieved to the endoplasmic reticulum. Using coatomer disassembly and epitope-specific
antibodies, we also demonstrate that the N- and C-terminal domains of β-COP are buried within the native coatomer complex.
These results provide the first insights into how the 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 Grant 31-33546.92 from the Fonds National Suisse (to T. E. K.) and by the Canton of Geneva. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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:
- COPs
-
coat proteins
- ER
-
endoplasmic reticulum
- ARF
-
ADP-ribosylation factor
- IP
-
immunoprecipitation
- GTP
S -
guanosine 5′-3-O-(thio)triphosphate
- PAGE
-
polyacrylamide gel electrophoresis
- BSA
-
bovine serum albumin.
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↵(2) J. Scheel, R. Pepperkok, M. Lowe, G. Griffiths and T. E. Kreis, manuscript in preparation.
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- Received February 10, 1995.
- Revision received October 10, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
