The vacuolar (H + )-ATPases are ATP-dependent proton pumps that function to acidify intracellular compartments and, in some cases, transport protons across the plasma membrane of eukaryotic cells. Intracellular V-ATPases play an important role in such normal physiological processes as receptor-mediated endocytosis, intracellular membrane traffic, pro-hormone processing, protein degradation and the coupled uptake of small molecules, such as neurotransmitters. They also function in the entry of various pathogenic agents, including many envelope viruses, like influenza virus, and toxins, like anthrax toxin. Plasma membrane V-ATPases function in renal pH homeostasis, bone resorption and sperm maturation, as well as in various disease processes, including renal tubular acidosis, osteopetrosis and tumor metastasis. V-ATPases are composed of a peripheral V 1 domain containing eight different subunits that is responsible for ATP hydrolysis and an integral V 0 domain containing six different subunits that translocates protons. In mammalian cells most of the V-ATPase subunits exist in multiple isoforms which are often expressed in a tissue specific manner. Isoforms of one of the V 0 subunits (subunit a) have been shown to possess information that targets the V-ATPase to distinct cellular destinations. Mutations in isoforms of subunit a lead to the human diseases osteopetrosis and renal tubular acidosis. A number of mechanisms are employed to regulate V-ATPase activity in vivo, including reversible dissociation of the V 1 and V 0 domains, control of the tightness of coupling of proton transport and ATP hydrolysis and selective targeting of V-ATPases to distinct cellular membranes. Isoforms of subunit a are involved in regulation both by control of coupling and by selective targeting. This review will begin with a brief introduction to the function, structure and mechanism of the VATPases followed by a discussion of the role of V-ATPase subunit isoforms and the mechanisms involved in regulation of V-ATPase activity. V-ATPase FunctionThe vacuolar (H + )-ATPases (V-ATPases) are a family of ATP-dependent proton pumps localized to a variety of cellular membranes of eukaryotic cells, including endosomes, lysosomes, Golgi-derived vesicles, secretory vesicles and, for some cells, the plasma membrane (1-3). V-ATPases within endosomes function to dissociate internalized ligands, such as low density lipoprotein (LDL), from their receptors, thus facilitating recycling of the receptors to the plasma membrane (4). They are also required for budding of endosomal carrier vesicles that carry internalized ligands from early to late endosomes (5) and for dissociation of lysosomal enzymes from the mannose-6-phosphate receptors that target them from the Golgi to the lysosome (6). Acidic endosomal compartments also provide the entry point for the cytotoxic portions of many envelope viruses, including influenza and Ebola virus, and toxins, such as diphtheria and anthrax toxin (7). The ability of these pathogenic agents to infect or kill ...

There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A(1)A(O) together with the bacterial F(1)F(O) ATP synthase. To address a potential role of the bacterial F(1)F(O) ATP synthase, we have exchanged the F(1)F(O) ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F(1)F(O) ATP synthase is dispensable for the growth of M. acetivorans.

N(epsilon)-acetyl-beta-lysine is a unique compatible solute found in methanogenic archaea grown at high salinities. Deletion of the genes that encode the lysine-2,3-aminomutase (ablA) and the beta-lysine acetyltransferase (ablB) abolished the production of N(epsilon)-acetyl-beta-lysine in Methanosarcina mazei Gö1. The mutant grew well at low and intermediate salinities. Interestingly, growth at high salt (800 mM NaCl) was only slowed down but not impaired demonstrating that in M. mazei Gö1 N(epsilon)-acetyl-beta-lysine is not essential for growth at high salinities. Nuclear magnetic resonance (NMR) analysis revealed an increased glutamate pool in the mutant. In addition to alpha-glutamate, a novel solute, alanine, was produced. The intracellular alanine concentration was as high as 0.36 +/- 0.05 micromol (mg protein)-1 representing up to 18% of the total solute pool at 800 mM NaCl. The cellular alanine concentration increased with the salinity of the medium and decreased in the presence of glycine betaine in the medium, indicating that alanine is used as compatible solute by M. mazei Gö1.

The methanogenic archaeon Methanosarcina mazei Gö1 accumulates glycine betaine in response to hypersalinity but differs from most other methanoarchaea in having two gene clusters both encoding a potential glycine betaine transporter, Ota and Otb. We have created mutants with either ota or otb deleted to address their role in salt adaptation. The mutants were not impaired in growth at low or high salt, neither at 37 degrees C nor at lower temperatures. However, the Deltaota mutant was completely defective in glycine betaine transport demonstrating that Ota is the only glycine betaine transporter in M. mazei. The mutation in otb led to increased transcription of ota and thus increased transport and accumulation of glycine betaine suggesting a cross talk between the two transporters.