The catalytic nucleotide binding subunit (subunit A) of the vacuolar proton-translocating ATPase (or V-ATPase) is homologous to the ␤-subunit of the F-ATPase but contains a 90-amino acid insert not present in the ␤-subunit, termed the nonhomologous region. We previously demonstrated that mutations in this region lead to changes in coupling of proton transport and ATPase activity and to inhibition of in vivo dissociation of the V-ATPase complex, an important regulatory mechanism (Shao, E., Nishi T., Kawasaki-Nishi, S., and The vacuolar proton-translocating ATPases (V-ATPases) 1 are a family of ATP-dependent proton pumps that couple the hydrolysis of ATP to proton movement across the membrane (1-8). This proton movement results in acidification of intracellular compartments, which in turn is critical for cellular processes such as receptor-mediated endocytosis, the processing and degradation of macromolecules, intracellular trafficking of lysosomal enzymes, coupled transport of small molecules, and entry of certain envelope viruses (1). For certain specialized cells such as renal intercalated cells, osteoclasts, macrophages, and insect goblet cells, V-ATPases are present on the plasma membrane and function in processes such as renal acidification, bone resorption, pH homeostasis, and coupled potassium transport (9 -12).The V-ATPases are multisubunit complexes composed of two functional domains (1-8). The soluble V 1 domain is responsible for ATP hydrolysis and contains eight different subunits (subunits A-H) with molecular masses 70 -13 kDa. The integral V 0 domain is responsible for proton translocation and contains six different subunits (subunits a, d, e, c, cЈ, and cЉ) with molecular masses of 100 -10 kDa. The 10-kDa subunit e, previously identified in bovine and insect (13,14), has recently been shown to be essential for function of the yeast V-ATPase (15). Both the 70-kDa A subunit and the 60-kDa B subunit of V 1 possess nucleotide binding sites (16,17), with the catalytic nucleotide binding sites located on subunit A (18). The V-ATPases structurally resemble the F-ATPases (proton-driven ATP synthases) of mitochondria, chloroplasts, and bacteria (19 -23). The A and B subunits of the V-ATPases share ϳ25% amino acid sequence identity with the ␤-and ␣-subunits of the F-ATPases, respectively (24 -28). Sequence alignment of the A subunit and the ␤-subunit reveals a 90-amino acid region (termed the nonhomologous domain), which is present in the A subunit but which is absent from the ␤-subunit (24 -27). Although not conserved between the V and F-ATPases, this region is highly conserved among V-ATPase A subunit sequences (24 -27).We have previously demonstrated by site-directed mutagenesis of the VMA1 gene that encodes subunit A in yeast that changes in the nonhomologous domain can alter coupling of proton transport and ATPase activity (29). We have also observed that mutations in this domain are able to block in vivo dissociation of the V-ATPase in response to glucose depletion (29). Reversible dissociation of the ...

Subunit A is the catalytic nucleotide binding subunit of the vacuolar proton-translocating ATPase (or V-ATPase) and is homologous to subunit ␤ of the F 1 F 0 ATP synthase (or F-ATPase). Amino acid sequence alignment of these subunits reveals a 90-amino acid insert in subunit A (termed the non-homologous region) that is absent from subunit ␤. To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1 gene that encodes subunit A in yeast. Substitutions were performed on 13 amino acid residues within this region that are conserved in all available A subunit sequences. Most of the 18 mutations introduced showed normal assembly of the V-ATPase. Of these, one (R219K) greatly reduced both proton transport and ATPase activity. By contrast, the P217V mutant showed significantly reduced ATPase activity but higher than normal levels of proton transport, suggesting an increase in coupling efficiency. Two other mutations in the same region (P223V and P233V) showed decreased coupling efficiency, suggesting that changes in the non-homologous region can alter coupling of proton transport and ATP hydrolysis. It was previously shown that the V-ATPase must possess at least 5-10% activity relative to wild type to undergo in vivo dissociation in response to glucose withdrawal. However, four of the mutations studied (G150A, D157E, P177V, and P223V) were partially or completely blocked in dissociation despite having greater than 30% of wild type levels of activity. These results suggest that changes in the non-homologous region can also alter in vivo dissociation of the V-ATPase independent of effects on activity.The vacuolar proton-translocating ATPases (or V-ATPases) 1 are ATP-driven proton pumps present in both intracellular compartments and the plasma membrane of eukaryotic cells (1-8). They couple the energy released upon ATP hydrolysis to the active transport of protons from the cytoplasm to either the lumen of various intracellular compartments or to the extracellular environment. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking of lysosomal enzymes, degradation of macromolecules, uptake of neurotransmitters, and the entry of various envelope viruses and toxins (1-8). Plasma membrane V-ATPases have also been implicated in many normal and disease processes, including bone resorption, renal acidification, pH homeostasis, and tumor metastasis (9 -13). Defects in specific V-ATPase subunits have been shown to be responsible for a number of human genetic diseases, including autosomal recessive osteopetrosis and renal tubular acidosis (9, 14 -16).The V-ATPases are multi-subunit complexes composed of two functional domains (1-8). The 640-kDa peripheral V 1 domain is responsible of ATP hydrolysis and consists of 8 different subunits (subunits A-H) with molecular masses of 70 -14 kDa. The V 0 domain is a 260-kDa integral complex composed of five different subunits (subunits a, d, cЈЈ, cЈ, and c with mole...

The vacuolar (H(+))-ATPases (or V-ATPases) are ATP-dependent proton pumps that function to acidify intracellular compartments in eukaryotic cells. This acidification is essential for such processes as receptor-mediated endocytosis, intracellular targeting of lysosomal enzymes, protein processing and degradation and the coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in such processes as renal acidification, bone resorption and pH homeostasis. Work from our laboratory has focused on the V-ATPases from clathrin-coated vesicles and yeast vacuoles.Structurally, the V-ATPases are composed of two domains: a peripheral complex (V(1)) composed of eight different subunits (A-H) that is responsible for ATP hydrolysis and an integral complex (V(0)) composed of five different subunits (a, d, c, c' and c") that is responsible for proton translocation. Electron microscopy has revealed the presence of multiple stalks connecting the V(1) and V(0) domains, and crosslinking has been used to address the arrangement of subunits in the complex. Site-directed mutagenesis has been employed to identify residues involved in ATP hydrolysis and proton translocation and to study the topology of the 100 kDa a subunit. This subunit has been shown to control intracellular targeting of the V-ATPase and to influence reversible dissociation and coupling of proton transport and ATP hydrolysis.

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Originally published in: Handbook of ATPases. Edited by Masamitsu Futai, Yoh Wada and Jack H. Kaplan. Copyright © 2004 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30689‐3 The sections in this article are Introduction Function of V ‐ ATP ases Overall Structure of V ‐ ATP ases and Relationship to F ‐ ATP ases Structure and Function of the Nucleotide‐binding Subunits Structure and Function of other V 1 Subunits and Arrangement of Subunits in the V ‐ ATP ase Complex Structure and Function of V 0 Subunits Mechanism of ATP ‐dependent Proton Transport Regulation of V ‐ ATP ase Activity In Vivo Conclusions Acknowledgments