STRUCTURE AND FUNCTION OF DYNAMIN, A 100KD GTPASE INVOLVED IN ENDOCYTOSIS

Diabetes, Digestive, Kidney Diseases

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Abstract

Dynamin, a 100 kDa GTPase, is believed to be involved in the constriction of clathrin coated pits and may cause fission of clathrin coated vesicles during receptor mediated endocytosis and during membrane retrieval in nerve terminals. We have shown 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. When GTP is added to the dynamin tubes they constrict and form small vesicles. These results provide strong evidence that dynamin is the structural component necessary for the formation of the constricted necks of coated pits, and support the hypothesis that dynamin is the force-generating molecule responsible for membrane fission. To further clarify the role of dynamin in membrane fission we have recently calculated a three-dimensional map of a dynamin mutant using cryo-electron microscopy and helical reconstruction methods. We found that dialyzing dynamin into GMP-PCP resulted in tubular crystals which diffract to approximately ~20 angstroms. Our STEM analysis of dynamin spirals treated with GMP-PCP indicates there are approximately 30 dynamin molecules per helical turn. The tubular crystals are similar in size and helical repeat to the constricted tubes seen when dynamin is treated with GTP. By helical processing methods we have calculated a 3D map of the dynamin tubes and found that the dynamin molecules extend out from the lipid bilayer in a T-shape dimer that can be divided into three distinct domains with the head and middle domains tightly interconnected between dynamin molecules. This strong interaction suggest a possible mechanism of tubular constriction which may lead to membrane fission in the cell. We are presently examining wild type and other dynamin mutants in different nucleotide states by this method. 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, to determine a common mechanism of action within this large family of GTPases.