No abstract

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 ZnCl 2 , 200 mM CaCl 2 , 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 V 1 subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.Eukaryotic cells contain an evolutionarily conserved enzyme, the vacuolar proton pump, V-ATPase 2 that couples the energy of ATP hydrolysis to proton transport across membranes. Intracellular V-ATPases are found in compartments such as clathrin-coated vesicles, Golgi, endosomes, lysosomes, secretory vesicles, and the central vacuoles of yeast as reviewed previously (1). V-ATPases are also present in the plasma membranes of specialized cells such as osteoclasts, renal intercalated cells, spermatids, neutrophils, and macrophages, where they function in such processes as bone resorption, renal acidification, pH homeostasis, and coupled transport (2-7). V-ATPases are complexes composed of at least 13 different subunits. These subunits are organized into two domains, a cytoplasmic V 1 domain that hydrolyzes ATP and an integral membrane V o domain that translocates protons across membranes. The V 1 is composed of eight subunits, A-H, with three copies of the nucleotide binding subunits A and B, and possibly two copies of subunit E, and two copies of subunit G (8, 9). The yeast V o is composed of six different subunits, a, c, cЈ, cЉ, d, and e, with four copies of subunit c (10 -13). Subunits a, c, cЈ, and cЉ are thought to be responsible for proton translocation, but the functions of subunits d (14) and e (10) are unknown. Proton translocation through V o is driven by rotational catalysis of V 1 (15).Yeast V-ATPases fa...