The Interaction of Calmodulin with Clathrin-coated Vesicles, Triskelions, and Light Chains
LOCALIZATION OF A BINDING SITE (*)
- Ursula M. Pley(1),
- Beth L. Hill(2),
- Christine Alibert(2),
- Frances M. Brodsky(3) and
- Peter Parham(1)(2)(§)
- From the (1)Departments of Microbiology and Immunology and
- (2)Cell Biology, Stanford University School of Medicine, Stanford, California 94305 and the
- (3)Department of Pharmacy and Pharmaceutical Chemistry, School of Pharmacy, and the Department of Microbiology and Immunology, School of Medicine, University of California, San Francisco, California 94143
- § To whom correspondence should be addressed: Dept. of Cell Biology, Fairchild Bldg., Stanford University, Stanford, CA 94305-5400. Tel.: 415-723-6224; Fax: 415-723-8464.
Abstract
The binding of clathrin-coated vesicles, clathrin triskelions, and free clathrin light chains to calmodulin-Sepharose was
compared. When isolated from bovine brain, all three components bound to calmodulin-Sepharose in the presence of calcium and
could be eluted by its removal. In contrast, coated vesicles and triskelions isolated from bovine adrenal gland did not bind
to calmodulin-Sepharose, although the free light chains from adrenal gland bound as effectively as those from brain. As distinct
isoforms of the clathrin light chains are expressed by brain and adrenal gland, these results implicate the clathrin light
chains as the calmodulin-binding component of coated vesicles and triskelions. Furthermore, the insertion sequences found
in the neuron-specific isoforms, although not necessary for the binding of free clathrin light chains to calmodulin, must
facilitate the interaction of heavy chain-associated light chains with calmodulin. Recombinant mutants of LCa, with deletions
spanning the entire sequence, were tested for binding to calmodulin-Sepharose. Those mutants retaining structural integrity,
as assessed by the binding of a panel of monoclonal antibodies, exhibited varying amounts of calmodulin binding activity.
However, deletion of the carboxyl-terminal 20 residues abolished calmodulin interaction. Thus, the carboxyl terminus of LCa
appears to constitute a calmodulin-binding site. Peptides corresponding to the carboxyl terminus of LCa or LCb inhibited the
interaction of the light chains with calmodulin, suggesting that this region forms the calmodulin-binding site of both LCa
and LCb. The carboxyl-terminal peptides of LCa and LCb inhibited the interaction of light chains with calmodulin 
10-fold less effectively than a calmodulin-binding peptide derived from smooth muscle myosin light chain kinase, but much
more effectively than a calmodulin-binding peptide derived from adenylate cyclase. This comparison places the clathrin light
chain-calmodulin interaction within the physiological range seen for other calmodulin-binding proteins.
Footnotes
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↵* This work was supported by grants from the American Cancer Society and the National Institutes of Health (to P. P.), by National Institutes of Health Grant GM38093 (to F. M. B.), by National Science Foundation Grant MCB-9118638, and by a grant from the Pew Charitable Trusts (to F. M. B.). 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:
- PAGE
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polyacrylamide gel electrophoresis
- MES
-
4-morpholineethanesulfonic acid.
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- Received May 9, 1994.
- Revision received November 7, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
