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J. Biol. Chem., Vol. 282, Issue 27, 19355-19364, July 6, 2007

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Mechanism of Dynamitin-mediated Disruption of Dynactin^*

From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

Dynamitin is a commonly used inhibitor of cytoplasmic dynein-based motility in living cells. Dynamitin does not inhibit dynein directly but instead acts by causing disassembly of dynactin, a multiprotein complex required for dynein-based movement. In dynactin, dynamitin is closely associated with the subunits p150^Glued and p24, which together form the shoulder and projecting arm structures of the dynactin molecule. In this study, we explore the way in which exogenous dynamitin effects dynactin disruption. We find that pure, recombinant dynamitin is an elongated protein with a strong propensity for self-assembly. Titration experiments reveal that free dynamitin binds dynactin before it causes release of subunits. When dynamitin is added to dynactin at an equimolar ratio of exogenous dynamitin subunits to endogenous dynamitin subunits (1x= 4 mol of exogenous dynamitin per mole of dynactin), exogenous dynamitin exchanges with endogenous dynamitin, and partial release of p150^Glued is observed. When added in vast excess (25x; 100 mol of exogenous dynamitin per mole of dynactin), recombinant dynamitin causes complete release of both p150^Glued subunits, two dynamitins and one p24, but not other dynactin subunits. Our data suggest that dynamitin mediates disruption of dynactin by binding to endogenous dynamitin subunits. This binding destabilizes the shoulder structure that links the p150^Glued arm to the Arp1 filament and leads to subunit release.

Received for publication, January 2, 2007 , and in revised form, April 12, 2007.

^* This work was supported in part by National Institutes of Health Grant GM44589 (to T. A. S.). 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.

¹ Supported by a fellowship from the J. D. French Alzheimers Foundation. Present address: Dept. of Biology, Long Island University, CW Post campus, Brookville, NY 11548.

² To whom correspondence should be addressed: Dept. of Biology, The Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218. Tel.: 410-516-5373; Fax: 410-516-5375; E-mail: schroer{at}jhu.edu.

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