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J. Biol. Chem., Vol. 279, Issue 39, 40431-40436, September 24, 2004

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Dynamin GTPase Domain Mutants That Differentially Affect GTP Binding, GTP Hydrolysis, and Clathrin-mediated Endocytosis^*

Byeong Doo Song, Marilyn Leonard, and Sandra L. Schmid

From the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

The GTPase dynamin is essential for clathrin-mediated endocytosis. Unlike most GTPases, dynamin has a low affinity for nucleotide, a high rate of GTP hydrolysis, and can self-assemble, forming higher order structures such as rings and spirals that exhibit up to 100-fold stimulated GTPase activity. The role(s) of GTP binding and/or hydrolysis in endocytosis remain unclear because mutations in the GTPase domain so far studied impair both. We generated a new series of GTPase domain mutants to probe the mechanism of GTP hydrolysis and to further test the role of GTP binding and/or hydrolysis in endocytosis. Each of the mutations had parallel effects on assembly-stimulated and basal GTPase activities. In contrast to previous reports, we find that mutation of Thr-65 to Ala (or Asp or His) dramatically lowered both the rate of assembly-stimulated GTP hydrolysis and the affinity for GTP. The assemblystimulated rate of hydrolysis was lowered by the mutation of Ser-61 to Asp and increased by the mutation of Thr-141 to Ala without significantly altering the K_m for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the K_m for GTP, suggesting that conformational changes in the active site accompany self-assembly. Analysis of transferrin endocytosis rates in cells overexpressing mutant dynamins revealed a stronger correlation with both the basal and assembly-stimulated rates of GTP hydrolysis than with the calculated ratio of dynamin-GTP/free dynamin, suggesting that GTP binding is not sufficient, and GTP hydrolysis is required for clathrin-mediated endocytosis in vivo.

Received for publication, June 23, 2004 , and in revised form, July 19, 2004.

^* This work was supported by National Institutes of Health Grant GM42455 (to S. L. S.). This is The Scripps Research Institute manuscript number 16557-CB. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 858-784-2311; Fax: 858-784-2345; E-mail: slschmid{at}scripps.edu.

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