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© The Rockefeller University Press,
0021-9525/2001//309 $5.00
The Journal of Cell Biology, Volume 152, Number 2,
, 2001 309-324
Original Article |
The Role of Dynamin and Its Binding Partners in Coated Pit Invagination and Scission
e.smythe{at}dundee.ac.uk
Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain–containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission.
To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.
We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.
Key Words: dynamin • amphiphysin • endophilin • synaptojanin • coated pits
© 2001 The Rockefeller University Press
Abbreviations used in this paper: B-SS-Tfn, biotinylated transferrin; GST; glutathione S-transferase; HEK, human embryonic kidney; PRD, proline/argine-rich domain; PtdInsP2, phosphoinositide 4,5-bisphosphate; SH, Src homology.
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