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SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Nature Cell Biology volume 1, pages 119–124 (1999)Cite this article

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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Figure 1: Cytosol- and ATP-dependent coated-vesicle formation is efficiently reconstituted in permeabilized 3T3-L1 cells.
Figure 2: Dynamin is necessary but not sufficient for coated-vesicle fission
Figure 3: GST–SH3 domains inhibit Tfn endocytosis in permeabilized 3T3-L1 cells.
Figure 4: Inhibition by GST–SH3 domains requires preincubation with membranes.
Figure 5: GST–SH3 domains inhibit distinct events in coated-vesicle formation.
Figure 6: Models for coated-vesicle formation in vitro.

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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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Authors and Affiliations

  1. Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines, La Jolla, 92037, California, USA

    Fiona Simpson & Sandra L. Schmid

  2. Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec , H3A 2B4, Canada

    Natasha K. Hussain & Peter S. McPherson

  3. Department of Biochemistry and Biophysics and the Hormone Research Institute, University of California, San Francisco, 94143-0534, California, USA

    Britta Qualmann & Regis B. Kelly

  4. Department of Pharmacology, University of Wisconsin, Madison, 53706-1532, Wisconsin, USA

    Brian K. Kay

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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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