Abstract
Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H+-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F0F1-type ATP synthase and the plasma membrane E1E2-type H+-ATPase. The vacuolar membrane H+-ATPase is composed of three major subunits, subunita (M r =67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H+-ATPase under two kinetic conditions have been determined to be 0.9–1.1×105 daltons for single-cycle hydrolysis of ATP and 4.1–5.3×105 daltons for multicycle hydrolysis of ATP, respectively.N,N′-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H+-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.
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Abbreviations
- CCCP:
-
carbonyl cyanidem-chlorophenyl hydrazone
- DCCD:
-
N,N′-dicyclohexylcarbondiimide
- DES:
-
diethylstilbestrol
- DIDS:
-
4,4′-diisothiocyano-2,2′-stilbene disulfonic acid
- NBD-Cl:
-
7-chloro-4-nitrobenzo-2-oxa-1,3-diazole
- Pi:
-
inorganic phosphate
- SDS:
-
sodium dodecylsulfate
- SF6847:
-
3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile
- SITS:
-
4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid
- ZW3-14:
-
N-tetradecyl-N,N′-dimethyl-3-ammonio-1-propanesulfonate
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Anraku, Y., Umemoto, N., Hirata, R. et al. Structure and function of the yeast vacuolar membrane proton ATPase. J Bioenerg Biomembr 21, 589–603 (1989). https://doi.org/10.1007/BF00808115
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DOI: https://doi.org/10.1007/BF00808115