The TFP1 gene of the yeast Saccharomyces cerevisiae encodes two proteins: 69-kilodalton (kD) catalytic subunit vacuolar proton-translocating adenosine triphosphatase (H + -ATPase) and a 50-kD protein. 69-kD is encoded by 5′ 3′ thirds coding region, whereas protein central third. Evidence presented that both proteins are obtained from single translation product cleaved to release spliced form subunit.

The vacuolar H+-ATPase of the yeast Saccharomyces cerevisiae is composed a complex peripheral subunits (the V1 sector) attached to an integral membrane V0 sector). In experiments described here, attachment sector was assessed in wild-type cells under variety growth conditions. Depriving glucose, even for as little 5 min, caused dissociation approximately 70% assembled enzyme complexes into separate and subcomplexes. Restoration glucose induced rapid efficient reassembly from previously...

The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. enzyme structurally organized into two major domains: peripheral domain (V1), containing the ATP binding sites, and an integral membrane (V0), forming proton pore. Dissociation of V1 V0 domains inhibits ATP-driven pumping, extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating extent association between domains. To examine mechanism this response, we...

Vacuolar acidification has been proposed to play a key role in number of cellular processes, including protein sorting, zymogen activation, and maintenance intracellular pH. We investigated the significance vacuolar by cloning mutagenizing gene for yeast proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying vat2 null allele were viable; however, these cells severely defective growth medium buffered at neutral Vacuoles isolated from bearing completely devoid activity. The...

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting both integral peripheral membrane subunits (Uchida, E., Ohsumi, Y., Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., Stevens, T. H. (1989) 264, 19236-19244). We obtained monoclonal antibodies recognizing 42- 100-kDa polypeptides were co-purified with activity. Using these we provide further...

Abstract The yeast vacuolar proton-translocating ATPase was isolated by two different methods. A previously reported purification of the enzyme (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095) repeated. This procedure consisted isolation vacuoles, solubilization with zwitterionic detergent ZW3-14, glycerol gradient centrifugation solubilized vacuoles. fraction highest specific activity (11 mumol ATP hydrolyzed mg-1 min-1) included eight polypeptides apparent...

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane V(o) domain. Multiple stresses induce changes in V1-V(o) assembly, but signaling mechanisms behind these not understood. Here we show that certain stress-responsive V-ATPase activity and assembly require lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). activation through response to salt...

Vacuolar proton-translocating ATPases are composed of a peripheral complex, V1, attached to an integral membrane Vo. Association the two complexes is essential for ATP-driven proton transport and regulated post-translationally in response glucose concentration. A new RAVE, was recently isolated implicated glucose-dependent reassembly V-ATPase that had disassembled deprivation (Seol, J. H., Shevchenko, A., Deshaies, R. (2001) Nat. Cell Biol. 3, 384–391). Here, we provide evidence supporting...

V-ATPases are composed of a peripheral complex containing the ATP-binding sites, V1 sector, attached to membrane proton pore, Vo sector. In vivo, free, inactive and sectors exist in dynamic equilibrium with fully assembled, active complexes, this can be perturbed by changes carbon source. Free complexes were isolated from cytosol wild-type yeast cells mutant strains lacking subunit c (Vma3p) or H (Vma13p). vma3Δ very similar, contained all previously identified subunits except C (Vma5p)....

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model intact V-ATPase calculated from two-dimensional projections complex at a resolution 25 angstroms. Images decorated with monoclonal antibodies against subunits A, E, G position within pseudo-hexagonal arrangement in V1, N terminus V1-V0 interface, C E top V1 domain. ESI...

Yeast mutants lacking vacuolar proton-translocating ATPase (V-ATPase) subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe, G. W., Fong, C. S., Alic, N., Higgins, V. J., and Dawes, I. W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6564–6569). We confirmed that V1 subunit (vma2Δ), Vo subunit, or either of the two isoforms are acutely H2O2 more menadione diamide than wild-type cells. The vma2Δ mutant contains elevated levels reactive oxygen...

ABSTRACT The yeast vacuolar H+ -ATPase (V-ATPase) consists of a complex peripheral subunits containing the ATP binding sites, termed V1 sector, attached to membrane proton pore, Vo sector. Interaction between and sectors is essential for ATP-driven transport, this interaction manipulated in vivo as means regulating V-ATPase activity. When (Saccharomyces cerevisiae) cells are deprived glucose little 5 min, up 75 % assembled complexes disassembled into cytoplasmic membrane-bound sectors....

Partial assembly of the peripheral and integral membrane sectors yeast vacuolar H(+)-ATPase has been detected in mutants lacking one subunit enzyme. Assembled complexes could be immunoprecipitated from biosynthetically labeled wild-type cells using monoclonal antibodies specific for 69- 60-kDa subunits enzyme, assembled (V0) a antibody against 100-kDa subunits. Parallel immunoprecipitations mutant revealed different degrees depending on that was missing. Partially also soluble, cytoplasmic...

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification basis these phenotypes, approximately 4700 deletion were screened defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 16 known subunits or assembly factors, 50 additional identified. Sixteen also grew poorly nonfermentable carbon sources, like mutants, analyzed further. The cwh36Delta mutant...

Vacuolar acidification has been proposed to play a key role in number of cellular processes, including protein sorting, zymogen activation, and maintenance intracellular pH. We investigated the significance vacuolar by cloning mutagenizing gene for yeast proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying vat2 null allele were viable; however, these cells severely defective growth medium buffered at neutral Vacuoles isolated from bearing completely devoid activity. The...