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J. Biol. Chem., Vol. 281, Issue 36, 26102-26111, September 8, 2006

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Effects of Human a3 and a4 Mutations That Result in Osteopetrosis and Distal Renal Tubular Acidosis on Yeast V-ATPase Expression and Activity^*

Noelle Ochotny, Aaron Van Vliet, Nelson Chan^¶, Yeqi Yao^¶, Mario Morel^¶, Norbert Kartner^¶, Herbert P. von Schroeder^¶, Johan N. M. Heersche^¶, and Morris F. Manolson^¶1

From the Department of Pharmacology, Department of Surgery, and ^¶Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada

V-ATPases are multimeric proton pumps. The 100-kDa "a" subunit is encoded by four isoforms (a1–a4) in mammals and two (Vph1p and Stv1p) in yeast. a3 is enriched in osteoclasts and is essential for bone resorption, whereas a4 is expressed in the distal nephron and acidifies urine. Mutations in human a3 and a4 result in osteopetrosis and distal renal tubular acidosis, respectively. Human a3 (G405R and R444L) and a4 (P524L and G820R) mutations were recreated in the yeast ortholog Vph1p, a3 (G424R and R462L), and a4 (W520L and G812R). Mutations in a3 resulted in wild type vacuolar acidification and growth on media containing 4 mM ZnCl₂, 200 mM CaCl₂, or buffered to pH 7.5 with V-ATPase hydrolytic and pumping activity decreased by 30–35%. Immunoblots confirmed wild type levels for V-ATPase a, A, and B subunits on vacuolar membranes. a4 G812R resulted in defective growth on selective media with V-ATPase hydrolytic and pumping activity decreased by 83–85% yet with wild type levels of a, A, and B subunits on vacuolar membranes. The a4 W520L mutation had defective growth on selective media with no detectable V-ATPase activity and reduced expression of a, A, and B subunits. The a4 W520L mutation phenotypes were dominant negative, as overexpression of wild type yeast a isoforms, Vph1p, or Stv1p, did not restore growth. However, deletion of endoplasmic reticulum assembly factors (Vma12p, Vma21p, and Vma22p) partially restored a and B expression. That a4 W520L affects both V_o and V₁ subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.

Received for publication, February 6, 2006 , and in revised form, June 27, 2006.

^* This work was supported by a Canadian Institutes of Health Research Operating Grant MOP-68952 and in part by a Canadian Foundation for Innovation/Ontario Innovation Trust Equipment Grant 7433 (to M. F. M.), and a graduate student scholarship from the Canadian Arthritis Network (to N. O.). 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: Faculty of Dentistry Research Institute, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G 1G6, Canada. Tel.: 416-979-4900 (ext. 4392); Fax: 416-979-4936; E-mail: m.manolson{at}utoronto.ca.

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