Structure And Function Of Dynamin, A 100kd Gtpase Involv

Diabetes, Digestive, Kidney Diseases

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Abstract

Dynamin, a 100 kDa GTPase, is essential for receptor mediated endocytosis, caveolae internalization and may play a role in vesicle trafficking in and out of the Golgi. We have shown previously that purified recombinant dynamin assembles into rings and spirals, and binds to lipid vesicles to form helical tubes which are similar to the dense material seen at the necks of clathrin-coated pits. Addition of GTP to the dynamin tubes results in constriction and fragmentation. These results strongly suggest dynamin is a mechanochemical enzyme localized to the necks of coated pits where it is responsible for membrane constriction and possibly membrane fission. To further clarify the role of dynamin in membrane fission we have calculated a three-dimensional map of a dynamin mutant (Delta-PRD) in the constricted state using cryo-electron microscopy and helical reconstruction methods. Our 23 Angstroms map reveals that dynamin exists as a dimer extending out from the lipid bilayer in a "T" shape that can be divided into three distinct densities; head, stalk and leg. We have docked the crystal structures of two domains into our 3D map: the PH domain of dynamin and the GTPase domain from the human guanylate binding protein 1 (hGBP1). The PH atomic structure fits extremely well into the leg density, the density that is slightly inserted into and extends out from the lipid bilayer. Similarly, the GTPase domain fits well into the head density, the largest region of our map. The overall structure suggests a possible mechanism of tubular constriction that may ultimately lead to membrane fission in the cell. We are presently examining wild type and dynamin mutants in the non-constricted state as well as obtaining a higher resolution map of the constricted state. This work will help elucidate how dynamin molecules interact and change conformation upon GTP addition and ultimately provide clues to how dynamin is regulated in the cell during endocytosis and its precise role in membrane fission. We are also examining other members of the dynamin family of proteins such as MxA, a protein involved in fighting viral infection, and Drp1, a dynamin-related protein involved in mitochondria fission. This work will help determine if a common mechanism of action exists within this large family of GTPases.