The uncoating of clathrin-coated vesicles can be mediated in vitro by the ‘uncoating ATPase’ that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Athough we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.
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October 1996
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Research Article|
October 01 1996
Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins
Michael E CHEETHAM;
Michael E CHEETHAM
*Department of Pathology, Institute of Ophthalmology, Bath Street, London, EC1V 9EL, U.K.
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Brian H. ANDERTON;
Brian H. ANDERTON
†Department of Neuroscience, Institute of Psychiatry, De Crespingy Park, London SE5 8AF, U.K.
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Antony P. JACKSON
Antony P. JACKSON
§
‡Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.
§To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
January 11 1996
Revision Received:
April 18 1996
Accepted:
June 10 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 319 (1): 103–108.
Article history
Received:
January 11 1996
Revision Received:
April 18 1996
Accepted:
June 10 1996
Citation
Michael E CHEETHAM, Brian H. ANDERTON, Antony P. JACKSON; Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins. Biochem J 1 October 1996; 319 (1): 103–108. doi: https://doi.org/10.1042/bj3190103
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