Abstract
Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.
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Acknowledgements
We thank J. Philie, E. De Heuvel and M. Yamabhai for providing reagents; M. Jost for critical reading of the manuscript; and W. Sossin for advice and support (to P.S.M.). The work was supported by grants from the NIH (S.L.S, R.B.K.) and the Natural Sciences and Engineering Research Council (P.S.M.) and by the University of Wisconsin Madison Medical School (B.K.K.). F.S. is supported by a Wellcome Trust Fellowship and B.Q by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. S.L.S. is an Established Investigator of the American Heart Association and P.S.M. is a Scholar of the Medical Research Council of Canada and an Alfred P. Sloan Research Fellow.
Correspondence and requests for materials should be addressed to P.S.M or S.L.S.
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Simpson, F., Hussain, N., Qualmann, B. et al. SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation. Nat Cell Biol 1, 119–124 (1999). https://doi.org/10.1038/10091
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DOI: https://doi.org/10.1038/10091
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