Zuo Jian, Vergara Sandra, Kohno Shinya, Holliday L Shannon
Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610, USA.
J Exp Biol. 2008 Apr;211(Pt 7):1102-8. doi: 10.1242/jeb.013672.
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 Kd 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.
液泡H⁺-ATP酶（V-ATP酶）是真核细胞中一种极其重要的酶，负责内吞区室的酸化。已证明来自哺乳动物和烟草天蛾的V-ATP酶的B亚基可结合肌动蛋白丝。破骨细胞的质膜靶向V-ATP酶需要B亚基的肌动蛋白结合活性。来自酿酒酵母的细菌表达的B亚基以195 nmol l⁻¹的解离常数（Kd）结合肌动蛋白丝。B亚基的肌动蛋白结合结构域因降低或消除肌动蛋白结合活性的突变而改变。当在无B亚基的酵母中表达时，突变体与内源性酵母亚基正确组装，巴弗洛霉素敏感的ATP酶活性与用野生型B亚基转化的酵母没有显著差异。含有突变亚基的酵母在pH 7.5时的生长情况与野生型一样好。用亚致死剂量的各种药物筛选无B亚基的酵母或用野生型或突变B亚基转化的无B亚基酵母，结果显示含有突变B亚基的酵母比用野生型B亚基转化的酵母对环己酰亚胺和渥曼青霉素更敏感。这些结果表明，肌动蛋白结合活性赋予酵母B亚基一种与其在质子泵酶活性中的作用不同的功能。
