The molecular chaperone calnexin associates with the vacuolar H(+)-ATPase from oat seedlings.

Acidification of endomembrane compartments by the vacuolar-type H(+)-ATPase (V-ATPase) is central to many cellular processes in eukaryotes, including osmoregulation and protein sorting. The V-ATPase complex consists of a peripheral sector (V1) and a membrane integral sector (V0); however, it is unclear how the multimeric enzyme is assembled. A 64-kD polypeptide that had copurified with oat V-ATPase subunits has been identified as calnexin, an integral protein on the endoplasmic reticulum. To determine whether calnexin interacted physically with the V-ATPase, microsomal membranes were Triton X-100 solubilized, and the protein-protein interaction was analyzed by coimmunoprecipitation. Monoclonal antibodies against calnexin precipitated both calnexin and V-ATPase subunits, including A and B and those of 44, 42, 36, 16, and 13 kD. A monoclonal antibody against subunit A precipitated the entire V-ATPase complex as well as calnexin and BiP, an endoplasmic reticulum lumen chaperone. The results support our hypothesis that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V1V0-ATPases at the endoplasmic reticulum.     

A 100 kDa polypeptide associates with the V0 membrane sector but not with the active oat vacuolar H(+)-ATPase, suggesting a role in assembly

The vacuolar H(+)-ATPase (V-ATPase) is responsible for acidifying endomembrane compartments in eukaryotic cells. Although a 100 kDa subunit is common to many V-ATPases, it is not detected in a purified and active pump from oat (Ward J.M. and Sze H. (1992) Plant Physiol. 99, 925-931). A 100 kDa subunit of the yeast V-ATPase is encoded by VPH1. Immunostaining revealed a Vph1p-related polypeptide in oat membranes, thus the role of this polypeptide was investigated. Membrane proteins were detergent-solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100 kDa polypeptide was not associated with the fully assembled ATPase; however, it was part of an approximately 250 kDa V0 complex including subunits of 36 and 16 kDa. Immunostaining with an affinity-purified antibody against the oat 100 kDa protein confirmed that the polypeptide was part of a 250 kDa complex and that it had not degraded in the approximately 670 kDa holoenzyme. Co-immunoprecipitation with a monoclonal antibody against A subunit indicated that peripheral subunits exist as assembled V1 subcomplexes in the cytosol. The free V1 subcomplex became attached to the detergent-solubilized V0 sector after mixing, as subunits of both sectors were co-precipitated by an antibody against subunit A. The absence of this polypeptide from the active enzyme suggests that, unlike the yeast Vph1p, the 100 kDa polypeptide in oat is not required for activity. Its association with the free Vo subcomplex would support a role of this protein in V-ATPase assembly and perhaps in sorting.     

Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V₀) and a peripherally associated catalytic sector (V₁). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V₀ sector as well as for V₁ attachment. Vma6p has been characterized as a nonintegrally associated V₀ subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.     

Structure, function and regulation of the vacuolar (H)-ATPases

The vacuolar (H⁺)-ATPases (or V-ATPases) function to acidify intracellular compartments in eukaryotic cells, playing an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, protein degradation and coupled transport. V-ATPases in the plasma membrane of specialized cells also function in renal acidification, bone resorption and cytosolic pH maintenance. The V-ATPases are composed of two domains. The V₁ domain is a 570-kDa peripheral complex composed of 8 subunits (subunits A–H) of molecular weight 70–13 kDa which is responsible for ATP hydrolysis. The V₀ domain is a 260-kDa integral complex composed of 5 subunits (subunits a–d) which is responsible for proton translocation. The V-ATPases are structurally related to the F-ATPases which function in ATP synthesis. Biochemical and mutational studies have begun to reveal the function of individual subunits and residues in V-ATPase activity. A central question in this field is the mechanism of regulation of vacuolar acidification in vivo. Evidence has been obtained suggesting a number of possible mechanisms of regulating V-ATPase activity, including reversible dissociation of V₁ and V₀ domains, disulfide bond formation at the catalytic site and differential targeting of V-ATPases. Control of anion conductance may also function to regulate vacuolar pH. Because of the diversity of functions of V-ATPases, cells most likely employ multiple mechanisms for controlling their activity.     

Structure,subunit function and regulation of the coated vesicle and yeast vacuolar (H(+))-ATPases

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.     

Mutational analysis of the subunit C (Vma5p) of the yeast vacuolar H+-ATPase.

Subunit C is a V(1) sector subunit found in all vacuolar H(+)-ATPases (V-ATPases) that may be part of the peripheral stalk connecting the peripheral V(1) sector with the membrane-bound V(0) sector of the enzyme (Wilkens, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804--31810). To elucidate subunit C function, we performed random and site-directed mutagenesis of the yeast VMA5 gene. Site-directed mutations in the most highly conserved region of Vma5p, residues 305--325, decreased catalytic activity of the V-ATPase by up to 48% without affecting assembly. A truncation mutant (K360stop) identified by random mutagenesis suggested a small region near the C terminus of the protein (amino acids 382--388) might be important for subunit stability. Site-directed mutagenesis revealed that three aromatic amino acids in this region (Tyr-382, Phe-385, and Tyr-388) in addition to four other conserved aromatic amino acids (Phe-260, Tyr-262, Phe-296, Phe-300) are essential for stable assembly of V(1) with V(0), although alanine substitutions at these positions support some activity in vivo. Surprisingly, three mutations (F260A, Y262A, and F385A) greatly decrease the stability of the V-ATPase in vitro but increase its k(cat) for ATP hydrolysis and proton transport by at least 3-fold. The peripheral stalk of V-ATPases must balance the stability essential for productive catalysis with the dynamic instability involved in regulation; these three mutations may perturb that balance.     

Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases

We identify a new naturally occurring class of inhibitor of vacuolar H+-ATPases (V-ATPases) isolated from vacuolar membranes of Neurospora crassa and from chromaffin granule membranes of Bos taurus. To date, the new class includes six chondropsins and poecillastrin A, large polyketide-derived macrolide lactams with 33-37 membered rings. In the National Cancer Institute's 60-cell screen the chondropsin class showed a tumor cell growth inhibitory fingerprint essentially indistinguishable from that of the bafilomycin/concanamycin and the salicylihalamide/lobatamide classes of well-established V-ATPase inhibitors. Half-maximal inhibition of V-ATPase activity in vitro occurred at 0.04-0.7 microM for the fungal vacuolar V-ATPase and at 0.4 to >10 microM for the chromaffin granule V-ATPase. Thus, the new inhibitors are somewhat less potent than the other two classes, which typically have Ki values of <10 nM for V-ATPases, and the new inhibitors differ from the other two classes in their specificity. The bafilomycin class inhibits all eucaryotic V-ATPases, the salicylihalamide class inhibits mammalian V-ATPases but not fungal V-ATPases, and the new chondropsin class inhibits the N. crassa V-ATPase better than the chromaffin granule V-ATPase. Two mutations in the N. crassa V-ATPase that affect the binding of bafilomycin had small but reproducible effects on the affinity of chondropsins for the V-ATPase, suggesting the possibility of a similar mechanism of inhibition.     

Function and subunit interactions of the N-terminal domain of subunit a (Vph1p) of the yeast V-ATPase

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0.     

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 vacuolar (H+)-ATPases--nature's most versatile proton pumps

The pH of intracellular compartments in eukaryotic cells is a carefully controlled parameter that affects many cellular processes, including intracellular membrane transport, prohormone processing and transport of neurotransmitters, as well as the entry of many viruses into cells. The transporters responsible for controlling this crucial parameter in many intracellular compartments are the vacuolar (H+)-ATPases (V-ATPases). Recent advances in our understanding of the structure and regulation of the V-ATPases, together with the mapping of human genetic defects to genes that encode V-ATPase subunits, have led to tremendous excitement in this field.     

Defective assembly of a hybrid vacuolar H(+)-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V(o) and catalytic V(1) sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 degrees C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 degrees C. Corresponding to the reversible defect of the hybrid V-ATPase, the V(o) subunit a epitope was exposed to the corresponding antibody at 37 degrees C, but became inaccessible at 30 degrees C. However, the V(1) sector was still associated with V(o) at 37 degrees C, as shown immunochemically. The control yeast V-ATPase was active at 37 degrees C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V(1) from V(o) in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex.

The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V(1)V(0) complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V(1) domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V(0) domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed.     

Proton translocation driven by ATP hydrolysis in V-ATPases

The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are multisubunit complexes composed of a peripheral domain (V(1)) responsible for ATP hydrolysis and an integral domain (V(0)) responsible for proton translocation. Based upon their structural similarity to the F(1)F(0) ATP synthases, the V-ATPases are thought to operate by a rotary mechanism in which ATP hydrolysis in V(1) drives rotation of a ring of proteolipid subunits in V(0). This review is focused on the current structural knowledge of the V-ATPases as it relates to the mechanism of ATP-driven proton translocation.     

Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain

The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.     

The amino-terminal domain of the E subunit of vacuolar H(+)-ATPase (V-ATPase) interacts with the H subunit and is required for V-ATPase function

Vacuolar H(+)-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F(1)F(0)-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A(1)-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.     

Saccharomyces cerevisiae Vacuolar H+-ATPase Regulation by Disassembly and Reassembly: One Structure and Multiple Signals

Vacuolar H⁺-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved.     

Effects of human a3 and a4 mutations that result in osteopetrosis and distal renal tubular acidosis on yeast V-ATPase expression and activity

V-ATPases are multimeric proton pumps. The 100-kDa "a" subunit is encoded by four isoforms (a1-a4) in mammals and two (Vph1p and Stv1p) in yeast. a3 is enriched in osteoclasts and is essential for bone resorption, whereas a4 is expressed in the distal nephron and acidifies urine. Mutations in human a3 and a4 result in osteopetrosis and distal renal tubular acidosis, respectively. Human a3 (G405R and R444L) and a4 (P524L and G820R) mutations were recreated in the yeast ortholog Vph1p, a3 (G424R and R462L), and a4 (W520L and G812R). Mutations in a3 resulted in wild type vacuolar acidification and growth on media containing 4 mM ZnCl2, 200 mM CaCl2, or buffered to pH 7.5 with V-ATPase hydrolytic and pumping activity decreased by 30-35%. Immunoblots confirmed wild type levels for V-ATPase a, A, and B subunits on vacuolar membranes. a4 G812R resulted in defective growth on selective media with V-ATPase hydrolytic and pumping activity decreased by 83-85% yet with wild type levels of a, A, and B subunits on vacuolar membranes. The a4 W520L mutation had defective growth on selective media with no detectable V-ATPase activity and reduced expression of a, A, and B subunits. The a4 W520L mutation phenotypes were dominant negative, as overexpression of wild type yeast a isoforms, Vph1p, or Stv1p, did not restore growth. However, deletion of endoplasmic reticulum assembly factors (Vma12p, Vma21p, and Vma22p) partially restored a and B expression. That a4 W520L affects both Vo and V1 subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.     

Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots

The vacuolar H⁺-translocating ATPase (H⁺-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A₁, a specific vacuolar-type ATPase inhibitor. The purified oat H⁺-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H⁺-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H⁺-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H⁺-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H⁺-ATPase. These results demonstrate that the vacuolar H⁺-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H⁺-ATPases exist in plants.     

Novel vacuolar H+-ATPase complexes resulting from overproduction of Vma5p and Vma13p.

The vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex composed of two sectors: V(1), a peripheral membrane sector responsible for ATP hydrolysis, and V(0), an integral membrane sector that forms a proton pore. Vma5p and Vma13p are V(1) sector subunits that have been implicated in the structural and functional coupling of the V-ATPase. Cells overexpressing Vma5p and Vma13p demonstrate a classic Vma(-) growth phenotype. Closer biochemical examination of Vma13p-overproducing strains revealed a functionally uncoupled V-ATPase in vacuolar vesicles. The ATP hydrolysis rate was 72% of the wild-type rate; but there was no proton translocation, and two V(1) subunits (Vma4p and Vma8p) were present at lower levels. Vma5p overproduction moderately affected both V-ATPase activity and proton translocation without affecting enzyme assembly. High level overexpression of Vma5p and Vma13p was lethal even in wild-type cells. In the absence of an intact V(0) sector, overproduction of Vma5p and Vma13p had a more detrimental effect on growth than their deletion. Overproduced Vma5p associated with cytosolic V(1) complexes; this association may cause the lethality.