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First published online March 14, 2008
Journal of Experimental Biology 211, 1102-1108 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.013672

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Biochemical and functional characterization of the actin-binding activity of the B subunit of yeast vacuolar H⁺-ATPase

Jian Zuo¹, Sandra Vergara¹, Shinya Kohno² and L. Shannon Holliday^1,3,*

¹ Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610, USA
² Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University, Hiroshima, Japan
³ Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610, USA

^* Author for correspondence (e-mail: sholliday{at}dental.ufl.edu)

Accepted 21 January 2008

Vacuolar H⁺-ATPase (V-ATPase) is a fundamentally important enzyme in eukaryotic cells that is responsible for acidification of endocytic compartments. The B subunits of V-ATPases from mammals and tobacco hornworm have been shown to bind actin filaments. Actin-binding activity by the B subunit is required for targeting V-ATPases to the plasma membrane of osteoclasts. Bacterially expressed B subunit from the yeast Saccharomyces cerevisiae bound actin filaments with a K_d of 195 nmol l^–1. The actin-binding domain of the B subunit was altered by mutations that reduced or eliminated the actin-binding activity. Mutants assembled properly with endogenous yeast subunits when expressed in B subunit-null yeast and bafilomycin-sensitive ATPase activity was not significantly different from yeast transformed with wild-type B subunit. Yeast containing the mutant subunits grew as well at pH 7.5 as wild-type. Screening null yeast or null yeast transformed with wild-type or mutant B subunits with sub-lethal doses of various drugs revealed that yeast containing the mutant B subunits were more sensitive to cycloheximide and wortmannin than those transformed with wild-type B subunits. These results suggest that actin-binding activity confers on the B subunit of yeast a function that is distinct from its role in the enzymatic activity of the proton pump.

Key words: V-ATPase, F-actin, Saccharomyces cerevisiae, actin-binding protein, cycloheximide, wortmannin, PI 3-kinase