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J. Biol. Chem., Vol. 282, Issue 42, 30466-30475, October 19, 2007

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Stimulation of Actin Polymerization by Vacuoles via Cdc42p-dependent Signaling^*

Sabina Isgandarova¹, Lynden Jones¹, Daniel Forsberg, Ana Loncar, John Dawson, Kelly Tedrick, and Gary Eitzen²

From the Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.

Received for publication, May 18, 2007 , and in revised form, August 1, 2007.

^* This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) and The Alberta Heritage Foundation for Medical Research (AHFMR). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6 and Table S2.

¹ These authors contributed equally to this work.

² A CIHR New Investigator and AHFMR Scholar. To whom correspondence should be addressed: 5-14 Medical Science Bldg., University of Alberta, Edmonton Alberta T6G 2H7, Canada. Fax: 780-492-0450; E-mail: gary.eitzen{at}ualberta.ca.

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