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J. Biol. Chem., Vol. 280, Issue 43, 36494-36501, October 28, 2005

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A Mammalian Actin Substitution in Yeast Actin (H372R) Causes a Suppressible Mitochondria/Vacuole Phenotype^*

Melissa McKane1, Kuo-Kuang Wen1, Istvan R. Boldogh, Sharmilee Ramcharan, Liza A. Pon, and Peter A. Rubenstein2

From the Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242 and the Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032

To determine the reason for the inviability of Saccharomyces cerevisiae with skeletal muscle actin, we introduced into yeast actin the first variant muscle residue from the C-terminal end, H372R. Arg is also found at this position in non-yeast nonmuscle actins. The substitution caused retarded growth on glucose and an inability to use glycerol as a sole carbon source. The mitochondria were clumped and had lost their DNA, the vacuole appeared hypervesiculated, and the actin cytoskeleton became somewhat depolarized. Introduction of the second muscle actin-specific substitution, S365A, rescued these defects. Suppression was also achieved by introducing the four acidic N-terminal residues of muscle actin in place of the two found in yeast actin. The H372R substitution results in an increase in polymerization-dependent fluorescence of Cys-374 pyrene-labeled actin. H372R actin polymerizes slightly faster than wild-type (WT) actin. Yeast actin-related proteins 2 and 3 (Arp2/3) accelerates the polymerization of H372R actin to a much greater extent than WT actin. The two suppressors did not affect the rate of H372R actin polymerization in the absence of an Arp2/3 complex. In contrast, the S365A substitution dampened the rate of Arp2/3 complex-stimulated H372R actin polymerization, and the addition of the four acidic N-terminal residues caused this rate to decrease below that observed with WT actin in the presence of Arp2/3. Structural analysis of the mutations suggests the presence of stringent steric and ionic requirements for the bottom of actin subdomain 1 and also suggests that there is allosteric communication through subdomain 1 within the actin monomer between the N and C termini.

Received for publication, June 27, 2005 , and in revised form, August 17, 2005.

^* This work was supported in part by National Institutes of Health Grants GM33689 (to P. A. R.) and GM45735 and GM 66037 (to L. A. P.). 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 Fig. 1 showing additional examples of actin and mitochondrial staining wild-type and H372R mutant cells.

¹ Should be considered as equal first authors.

² To whom correspondence should be addressed. Tel.: 319-335-7911; E-mail: peter-rubenstein{at}uiowa.edu.

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