Biochemical Heterogeneity and Phosphorylation of Coatomer Subunits (*)
- From the Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002 and the Department of Cell Biology, University of Geneva, Sciences III, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
- ** To whom correspondence should be addressed: Dept. of Cell Biology, Yale University School of Medicine, 333 Cedar St., P.O. Box 208002, New Haven, CT 06520-8002. Tel.: 203-785-5058; Fax: 203-785-7226; Ira.Mellman{at}yale.edu.
Abstract
The coat protomer complex I (COPI) family of coat proteins are involved in the assembly of membrane-associated coats thought
to mediate vesicular transport between the endoplasmic reticulum and the Golgi complex, between adjacent Golgi cisternae,
and possibly in the endocytic pathway. We investigated whether this heterogeneity in the sites of COPI action might be reflected
in biochemical heterogeneity of one or more COPI subunits. A simplified method was devised to purify the cytosolic COPI precursor
complex, coatomer, from rat liver cytosol. The individual subunits were analyzed by high resolution two dimensional gel electrophoresis
and mass spectroscopic analysis of tryptic peptides. Considerable charge heterogeneity was observed, particularly for the
β-COP and 
-COP subunits. The multiple species detected, however, did not appear to reflect the presence of distinct translation products
but rather a significant degree of protein phosphorylation. The observed pI of β-COP was sensitive to alkaline phosphatase
digestion. Moreover, isolation of coatomer from metabolically labeled tissue culture cells demonstrated directly that both
β-COP and 
-COP, but no other coatomer subunits, were serine-phosphorylated. COPI phosphorylation may regulate coatomer assembly, membrane
recruitment, or the specificity of coatomer-organelle interaction.
Footnotes
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↵§ Supported by National Institutes of Health National Research Service Award Fellowship F32GM16339.
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↵¶ Supported by a Travelling Fellowship from the Wellcome Trust.
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↵* This work was supported in part by research grants from the National Institutes of Health (to I. M.) (GM29765), from the Fonds National Suisse (to T. E. K.) (31-33546.92), and from the Human Frontier Science Foundation (to I. M. and T. E. K.). 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:
- TGN
-
trans-Golgi network
- COP
-
coat protomer complex
- COPI and COPII
-
coat protomer complexes I and II, respectively
- ER
-
endoplasmic reticulum
- S100
-
100,000 × g supernatant
- PAGE
-
polyacrylamide gel electrophoresis
- IEF
-
isoelectric focusing
- CHO
-
Chinese hamster ovary
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- MEM
-
minimal essential medium.
-
↵2 M. Lu, M. Gomez, T. E. Kreis, and I. Mellman, unpublished data.
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↵3 M. Kim and I. Mellman, unpublished data.
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- Received August 4, 1995.
- Revision received January 9, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
