Arrangement of subunits in the proteolipid ring of the V-ATPase

The vacuolar ATPases (V-ATPases) are multisubunit complexes containing two domains. The V(1) domain (subunits A-H) is peripheral and carries out ATP hydrolysis. The V(0) domain (subunits a, c, c', c', d, and e) is membrane-integral and carries out proton transport. In yeast, there are three proteolipid subunits as follows: subunit c (Vma3p), subunit c' (Vma11p), and subunit c' (Vma16p). The proteolipid subunits form a six-membered ring containing single copies of subunits c' and c' and four copies of subunit c. To determine the possible arrangements of proteolipid subunits in V(0) that give rise to a functional V-ATPase complex, a series of gene fusions was constructed to constrain the arrangement of pairs of subunits in the ring. Fusions containing c' employed a truncated version of this protein lacking the first putative transmembrane helix (which we have shown previously to be functional), to ensure that the N and C termini of all subunits were located on the luminal side of the membrane. Fusion constructs were expressed in strains disrupted in c', c', or both but containing a wild copy of c to ensure the presence of the required number of copies of subunit c. The c-c'(DeltaTM1), c'(DeltaTM1)-c', and c'-c constructs all complemented the vma(-) phenotype and gave rise to complexes possessing greater than 25% of wild-type levels of activity. By contrast, neither the c-c', the c'-c'(DeltaTM1), nor the c'(DeltaTM1)-c constructs complemented the vma(-) phenotype. These results suggest that functionally assembled V-ATPase complexes contain the proteolipid subunits arranged in a unique order in the ring.     

TM2 but not TM4 of subunit c'' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking

The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c') relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c'. Subunit c' contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908-41913). We have now analyzed interaction of subunits a and c' using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c' centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c' does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.     

Definition of membrane topology and identification of residues important for transport in subunit a of the vacuolar ATPase

Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.     

Interacting helical surfaces of the transmembrane segments of subunits a and c' of the yeast V-ATPase defined by disulfide-mediated cross-linking

Proton translocation by the vacuolar (H+)-ATPase (or V-ATPase) has been shown by mutagenesis to be dependent upon charged residues present within transmembrane segments of subunit a as well as the three proteolipid subunits (c, c', and c"). Interaction between R735 in TM7 of subunit a and the glutamic acid residue in the middle of TM4 of subunits c and c' or TM2 of subunit c" has been proposed to be essential for proton release to the luminal compartment. In order to determine whether the helical face of TM7 of subunit a containing R735 is capable of interacting with the helical face of TM4 of subunit c' containing the essential glutamic acid residue (Glu-145), cysteine-mediated cross-linking between these subunits in yeast has been performed. Cys-less forms of subunits a and c' as well as forms containing unique cysteine residues were constructed, introduced together into a strain disrupted in both endogenous subunits, and tested for growth at neutral pH, for assembly competence and for cross-linking in the presence of cupric-phenanthroline by SDS-PAGE and Western blot analysis. Four different cysteine mutants of subunit a were each tested pairwise with ten different unique cysteine mutants of subunit c'. Strong cross-linking was observed for the pairs aS728C/c'I142C, aA731C/c'E145C, aA738C/c'F143C, aA738C/c'L147C, and aL739C/c'L147C. Partial cross-linking was observed for an additional 13 of 40 pairs analyzed. When arrayed on a helical wheel diagram, the results suggest that the helical face of TM7 of subunit a containing Arg-735 interacts with the helical face of TM4 of subunit c' centered on Val-146 and bounded by Glu-145 and Leu-147. The results are consistent with a possible rotational flexibility of one or both of these transmembrane segments as well as some flexibility of movement perpendicular to the membrane.     

Analysis of strains with mutations in six genes encoding subunits of the V-ATPase: eukaryotes differ in the composition of the V0 sector of the enzyme

To address questions about the structure of the vacuolar ATPase, we have generated mutant strains of Neurospora crassa defective in six subunits, C, H, a, c, c', and c'. Except for strains lacking subunit c', the mutant strains were indistinguishable from each other in most phenotypic characteristics. They did not accumulate arginine in the vacuoles, grew poorly at pH 5.8 with altered morphology, and failed to grow at alkaline pH. Consistent with findings from Saccharomyces cerevisiae, the data indicate that subunits C and H are essential for generation of a functional enzyme. Unlike S. cerevisiae, N. crassa has a single isoform of the a subunit. Analysis of other fungal genomes indicates that only the budding yeasts have a two-gene family for subunit a. It has been unclear whether subunit c', a small proteolipid, is a component of all V-ATPases. Our data suggest that this subunit is present in all fungi, but not in other organisms. Mutation or deletion of the N. crassa gene encoding subunit c' did not completely eliminate V-ATPase function. Unlike other V-ATPase null strains, they grew, although slowly, at alkaline pH, were able to form conidia (asexual spores), and were inhibited by concanamycin, a specific inhibitor of the V-ATPase. The phenotypic character in which strains differed was the ability to go through the sexual cycle to generate mature spores and viable mutant progeny. Strains lacking the integral membrane subunits a, c, c', and c' had more severe defects than strains lacking subunits C or H.     

Structure and regulation of the vacuolar ATPases

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for both acidification of intracellular compartments and, for certain cell types, proton transport across the plasma membrane. Intracellular V-ATPases function in both endocytic and intracellular membrane traffic, processing and degradation of macromolecules in secretory and digestive compartments, coupled transport of small molecules such as neurotransmitters and ATP and in the entry of pathogenic agents, including envelope viruses and bacterial toxins. V-ATPases are present in the plasma membrane of renal cells, osteoclasts, macrophages, epididymal cells and certain tumor cells where they are important for urinary acidification, bone resorption, pH homeostasis, sperm maturation and tumor cell invasion, respectively. The V-ATPases are composed of a peripheral domain (V(1)) that carries out ATP hydrolysis and an integral domain (V(0)) responsible for proton transport. V(1) contains eight subunits (A-H) while V(0) contains six subunits (a, c, c', c', d and e). V-ATPases operate by a rotary mechanism in which ATP hydrolysis within V(1) drives rotation of a central rotary domain, that includes a ring of proteolipid subunits (c, c' and c'), relative to the remainder of the complex. Rotation of the proteolipid ring relative to subunit a within V(0) drives active transport of protons across the membrane. Two important mechanisms of regulating V-ATPase activity in vivo are reversible dissociation of the V(1) and V(0) domains and changes in coupling efficiency of proton transport and ATP hydrolysis. This review focuses on recent advances in our lab in understanding the structure and regulation of the V-ATPases.     

The first putative transmembrane segment of subunit c" (Vma16p) of the yeast V-ATPase is not necessary for function

The yeast vacuolar ATPase (V-ATPase) contains three proteolipid subunits: c (Vma3p), c' (Vma11p), and c" (Vma16p). Each subunit contains a buried glutamate residue that is essential for function, and these subunits are not able to substitute for each other in supporting activity. Subunits c and c' each contain four putative transmembrane segments (TM1-4), whereas subunit c" is predicted to contain five. To determine whether TM1 of subunit c" serves an essential function, a deletion mutant of Vma16p was constructed lacking TM1 (Vma16p-Delta TM1). Although this construct does not complement the loss of Vma3p or Vma11p, it does complement the loss of full-length Vma16p. Vacuoles isolated from the strain expressing Vma16p-Delta TM1 showed V-ATPase activity and proton transport greater than 80% relative to wild type and displayed wild type levels of subunits A and a, suggesting normal assembly of the V-ATPase complex. These results suggest that TM1 of Vma16p is dispensable for both activity and assembly of the V-ATPase. To obtain information about the topology of Vma16p, labeling of single cysteine-containing mutants using the membrane-permeable reagent 3-(N-maleimidylpropionyl)biocytin (MPB) and the -impermeable reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) was tested. Both the Cys-less form of Vma16p and eight single cysteine-containing mutants retained greater than 80% of wild type levels of activity. Of the eight mutants tested, two (S5C and S178C) were labeled by MPB. MPB-labeling of S5C was blocked by AMS in intact vacuoles, whereas S178C was blocked by AMS only in the presence of permeabilizing concentrations of detergent. In addition, a hemagglutinin epitope tag introduced into the C terminus of Vma16p was recognized by an anti-hemagglutinin antibody in intact vacuolar membranes, suggesting a cytoplasmic orientation for the C terminus. These results suggest that subunit c" contains four rather than five transmembrane segments with both the N and C terminus on the cytoplasmic side of the membrane.     

Molecular characterization of XVSAP1, a stress-responsive gene from the resurrection plant Xerophyta viscosa Baker

The strategy of 'complementation by functional sufficiency' was used to isolate a cDNA designated XVSAP1 from a cDNA library constructed from dehydrated Xerophyta viscosa Baker leaves. Analysis of the cDNA sequence indicated a highly hydrophobic protein with six transmembrane regions. Southern blot analysis revealed that there are at least two copies of XVSAP1 in X. viscosa. The deduced amino acid sequence showed 49% identity to WCOR413, a low-temperature-regulated protein from wheat. The protein also showed between 25% to 56% identity to WCOR413-like proteins from Arabidopsis thaliana. Expression of XVSAP1 in Escherichia coli (srl::Tn10) conferred osmotic stress tolerance when the cells were grown in 1 M sorbitol. Analysis of gene expression using semi-quantitative RT-PCR indicated that XVSAP1 is induced by dehydration, salt stress (100 mM), both low (4 degrees C) and high temperature (42 degrees C) and high light treatment (1500 micromol m(-2) s(-1)). These results suggest that XVSAP1 may have a significant role to play in the response of X. viscosa to abiotic stresses.     

Interaction of inhibitors of the vacuolar H(+)-ATPase with the transmembrane Vo-sector

The macrolide antibiotic concanamycin A and a designed derivative of 5-(2-indolyl)-2,4-pentadienamide (INDOL0) are potent inhibitors of vacuolar H(+)-ATPases, with IC(50) values in the low and medium nanomolar range, respectively. Interaction of these V-ATPase inhibitors with spin-labeled subunit c in the transmembrane V(o)-sector of the ATPase was studied by using the transport-active 16-kDa proteolipid analogue of subunit c from the hepatopancreas of Nephrops norvegicus. Analogous experiments were also performed with vacuolar membranes from Saccharomyces cerevisiae. Membranous preparations of the Nephrops 16-kDa proteolipid were spin-labeled either on the unique cysteine C54, with a nitroxyl maleimide, or on the functionally essential glutamate E140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). These residues were previously demonstrated to be accessible to lipid. Interaction of the inhibitors with these lipid-exposed residues was studied by using both conventional and saturation transfer EPR spectroscopy. Immobilization of the spin-labeled residues by the inhibitors was observed on both the nanosecond and microsecond time scales. The perturbation by INDOL0 was mostly greater than that by concanamycin A. Qualitatively similar but quantitatively greater effects were obtained with the same spin-label reagents and vacuolar membranes in which the Nephrops 16-kDa proteolipid was expressed in place of the native vma3p proteolipid of yeast. The spin-label immobilization corresponds to a direct interaction of the inhibitors with these intramembranous sites on the protein. A mutational analysis on transmembrane segment 4 known to give resistance to concanamycin A also gave partial resistance to INDOL0. The results are consistent with transmembrane segments 2 and 4 of the 16-kDa putative four-helix bundle, and particularly the functionally essential protonation locus, being involved in the inhibitor binding sites. Inhibition of proton transport may also involve immobilization of the overall rotation of the proteolipid subunit assembly.     

Topological characterization of the c, c', and c" subunits of the vacuolar ATPase from the yeast Saccharomyces cerevisiae

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V(1) and V(0). Hydrolysis of ATP in the V(1) subcomplex is tightly coupled to proton translocation accomplished by the V(0) subcomplex, which is composed of five unique subunits (a, d, c, c', and c"). Three of the subunits, subunit c (Vma3p), c' (Vma11p), and c" (Vma16p), are small highly hydrophobic integral membrane proteins called "proteolipids" that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c' (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c" (Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c' have four transmembrane segments with their N and C termini in the lumen and that c" has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal.     

Incorporation of transmembrane peptides from the vacuolar H(+)-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy

Peptides were designed that are based on candidate transmembrane sequences of the V o-sector from the vacuolar H (+)-ATPase of Saccharomyces cerevisiae. Spin-label EPR studies of lipid-protein interactions were used to characterize the state of oligomerization, and polarized IR spectroscopy was used to determine the secondary structure and orientation, of these peptides in lipid bilayer membranes. Peptides corresponding to the second and fourth transmembrane domains (TM2 and TM4) of proteolipid subunit c (Vma3p) and of the putative seventh transmembrane domain (TM7) of subunit a (Vph1p) are wholly, or predominantly, alpha-helical in membranes of dioleoyl phosphatidylcholine. All three peptides self-assemble into oligomers of different sizes, in which the helices are differently inclined with respect to the membrane normal. The coassembly of rotor (Vma3p TM4) and stator (Vph1p TM7) peptides, which respectively contain the glutamate and arginine residues essential to proton transport by the rotary ATPase mechanism, is demonstrated from changes in the lipid interaction stoichiometry and helix orientation. Concanamycin, a potent V-ATPase inhibitor, and a 5-(2-indolyl)-2,4-pentadienoyl inhibitor that exhibits selectivity for the osteoclast subtype, interact with the membrane-incorporated Vma3p TM4 peptide, as evidenced by changes in helix orientation; concanamycin additionally interacts with Vph1p TM7, suggesting that both stator and rotor elements contribute to the inhibitor site within the membrane. Comparison of the peptide behavior in lipid bilayers is made with membranous subunit c assemblies of the 16-kDa proteolipid from Nephrops norvegicus, which can substitute functionally for Vma3p in S. cerevisiae.     

The Prokaryote-to-Eukaryote Transition Reflected in the Evolution of the V/F/A-ATPase Catalytic and Proteolipid Subunits

Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed. Received: 27 August 1997 / Accepted: 14 January 1998     

VMA11, a novel gene that encodes a putative proteolipid, is indispensable for expression of yeast vacuolar membrane H(+)-ATPase activity.

A gene, VMA11, is indispensable for expression of the vacuolar membrane H(+)-ATPase activity in the yeast Saccharomyces cerevisiae (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The VMA11 gene was isolated from a yeast genomic DNA library by complementation of the vma11 mutation. The nucleotide sequence of the gene predicts a hydrophobic proteolipid of 164 amino acids with a calculated molecular mass of 17,037 daltons. The deduced amino acid sequence shows 56.7% identity, and significant coincidence in amino acid composition with the 16-kDa subunit c (a VMA3 gene product) of the yeast vacuolar membrane H(+)-ATPase. VMA11 and VMA3 on a multicopy plasmid did not suppress the vma3 and vma11 mutation, respectively, suggesting functional independence of the two gene products. Biochemical detection of the VMA11 gene product was unsuccessful, but vacuoles in the VMA11-disrupted cells were not assembled with either subunit c or subunits a and b of the H(+)-ATPase, resulting in defects of the activity and in vivo vacuolar acidification.     

Identification of lipid-accessible sites on the nephrops 16-kDa proteolipid incorporated into a hybrid vacuolar H(+)-ATPase: site-directed labeling with N-(1-Pyrenyl)cyclohexylcarbodiimide and fluorescence quenching analysis

Proton translocation by the vacuolar H(+)-ATPase is mediated by a multicopy transmembrane protein, the 16-kDa proteolipid. It is proposed to assemble in the membrane as a hexameric complex, with each polypeptide comprising four transmembrane helices. The fourth helix of the proteolipid contains an intramembrane acidic residue (Glu140) which is essential for proton translocation and is reactive toward N,N'-dicyclohexylcarbodiimide (DCCD). Current theoretical models of proton translocation by the vacuolar ATPase require that Glu140 should be protonated and in contact with the membrane lipid. In this study we present direct support for this hypothesis. Modification with the fluorescent DCCD analogue N-(1-pyrenyl)cyclohexylcarbodiimide, coupled to fluorescence quenching studies and bilayer depth measurements using the parallax method, was used to probe the position of Glu140 with respect to the bilayer. Glutamate residues were also introduced mutagenically as targets for the fluorescent probe in order to map additional lipid-accessible sites on the 16-kDa proteolipid. These data are consistent with a structural model of the 16-kDa proteolipid oligomer in which the key functional residue Glu140 and discrete faces of the second and third transmembrane helices of the 16-kDa proteolipid are exposed at the lipid-protein interface.     

Interaction of spin-labeled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector

The osteoclast variant of the vacuolar H⁺-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC₅₀ in the 10-nanomolar range.     

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.     

Assembly of the yeast vacuolar H+-ATPase and ATP hydrolysis occurs in the absence of subunit c''

The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c' is not essential for assembly but is for functional coupling of the enzyme.