Hsc70 Chaperones Clathrin and Primes It to Interact with Vesicle Membranes*
- From the Laboratory of Cell Biology, Section on Cell Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
Abstract
When Hsc70 uncoats clathrin-coated vesicles in an auxilin- and ATP-dependent reaction, a single round of rapid uncoating occurs followed by very slow steady-state uncoating. We now show that this biphasic time course occurs because Hsc70 sequentially forms two types of complex with the dissociated clathrin triskelions. The first round of clathrin uncoating is driven by formation of a pre-steady-state assembly protein (AP)-clathrin-Hsc70-ADP complex. Then, following exchange of ADP with ATP, a steady-state AP-clathrin-Hsc70-ATP complex forms that ties up Hsc70, preventing further uncoating. This steady-state complex forms only during uncoating in the presence of APs; in the absence of APs, Hsc70 rapidly dissociates from the uncoated clathrin and continues to carry out uncoating. Whether it is complexed with ATP or ADP, the steady-state complex has very different properties from the pre-steady-state complex in that it cannot be immunoprecipitated by anti-clathrin antibodies and is readily dissociated by fast protein liquid chromatography. Remarkably, when the steady-state complex is incubated with uncoated vesicle membranes in ATP, the pre-steady-state complex reforms, suggesting that the clathrin triskelions in the steady-state complex rebind to the membranes and are again uncoated by Hsc70. We propose that Hsc70 not only uncoats clathrin but also chaperones it to prevent it from inappropriately polymerizing in the cell cytosol and primes it to reform clathrin-coated pits.
Footnotes
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↵* 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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↵‡ Present address: Dept. of Immunology, Jerome H. Holland Laboratory, American Red Cross, Rockville, MD 20855.
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↵§ These two authors contributed equally to this work.
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↵¶ Present address: Dept. of Physiology and Cell Biology MS-341, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208.
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↵‖ Present address: Dept. of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245.
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↵** To whom correspondence should be addressed: Laboratory of Cell Biology, National Institutes of Health, Bldg. 3, Rm. B1-22, Bethesda, MD 20892-0301, Tel.: 301-496-2846; Fax: 301-402-1519; E-mail: eisenbee@nhlbi.nih.gov.
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↵2 R. Jiang, B. Gao, K. Prasad, L. E. Greene, and E. Eisenberg, unpublished data.
- Abbreviations:
- AP
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assembly protein
- Hsp70
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70-kDa heat shock protein
- Hsc70
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the constitutive isoform of Hsp70
- FPLC
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fast protein liquid chromatography
- Mes
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2-(N-morpholino)ethanesulfonic acid
- His6-Hsp70
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Hsp70 constructed with six-histidine tag at its N terminus
- DTT
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dithiothreitol
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- Received October 19, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
