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J. Biol. Chem., Vol. 281, Issue 29, 20129-20139, July 21, 2006

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Effects of Human Deafness -Actin Mutations (DFNA20/26) on Actin Function^*

Keith E. Bryan, Kuo-Kuang Wen, Mei Zhu, Nanna Dahl Rendtorff^¶, Michael Feldkamp, Lisbeth Tranebjaerg^¶||, Karen H. Friderici, and Peter A. Rubenstein¹

From the Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, the Departments of Microbiology & Molecular Genetics and Pediatrics & Human Development, Michigan State University, East Lansing, Michigan 48824, the ^¶Wilhem Johannsen Centre for Functional Genomics, Department of Medical Biochemistry and Genetics, University of Copenhagen, DK-2400 Copenhagen K, Denmark, and the ^||Department of Audiology, Bispebjerg Hospital, 2400 Copenhagen NV, Denmark

Six point mutations in non-muscle -actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each -actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize.

Received for publication, February 16, 2006 , and in revised form, April 21, 2006.

^* This work was supported in part by National Institutes of Health Grants GM33689 (to P. A. R.) and DC004568 (to K. H. F.) and Oticon Foundation Grant 22-00-0678 (to L. T.). 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 To whom correspondence should be addressed: Dept. of Biochemistry, University of Iowa Carver College of Medicine, Bowen Science Bldg., 51 Newton Rd., Iowa City, IA 52242. Tel.: 319-335-7911; Fax: 319-335-9570; E-mail: peter-rubenstein{at}uiowa.edu.

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				K. E. Bryan and P. A. Rubenstein Allele-specific Effects of Human Deafness {gamma}-Actin Mutations (DFNA20/26) on the Actin/Cofilin Interaction J. Biol. Chem., July 3, 2009; 284(27): 18260 - 18269. [Abstract] [Full Text] [PDF]

				E. Stokasimov, M. McKane, and P. A. Rubenstein Role of Intermonomer Ionic Bridges in the Stabilization of the Actin Filament J. Biol. Chem., December 12, 2008; 283(50): 34844 - 34854. [Abstract] [Full Text] [PDF]