Functional characterization of the N-terminal domain of subunit H (Vma13p) of the yeast vacuolar ATPase

The yeast Saccharomyces cerevisiae vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex responsible for acidifying intracellular organelles and is highly regulated. One of the regulatory subunits, subunit H, is encoded by the VMA13 gene in yeast and is composed of two domains, the N-terminal domain (amino acids (aa) 1-352) and the C-terminal domain (aa 353-478). The N-terminal domain is required for the activation of the complex, whereas the C-terminal domain is required for coupling ATP hydrolysis to proton translocation (Liu, M., Tarsio, M., Charsky, C. M., and Kane, P. M. (2005) J. Biol. Chem. 280, 36978-36985). Experiments with epitope-tagged copies of Vma13p revealed that there is only one copy of Vma13p/subunit H per V-ATPase complex. Analysis of the N-terminal domain shows that the first 179 amino acids are not required for the activation and full function of the V-ATPase complex and that the minimal region of Vma13p/subunit H capable of activating the V-ATPase is aa 180-353 of the N-terminal domain. Subunit H is expressed as two splice variants in mammals, and deletion of 18 amino acids in yeast Vma13p corresponding to the mammalian subunit H beta isoform results in reduced V-ATPase activity and significantly lower coupling of ATPase hydrolysis to proton translocation. Intriguingly, the yeast Vma13p mimicking the mammalian subunit H beta isoform is functionally equivalent to Vma13p lacking the entire C-terminal domain. These results suggest that the mammalian V-ATPase complexes with subunit H splice variant SFD-alpha or SFD-beta are likely to have different activities and may perform distinct cellular functions.     

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.     

Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H(+)-ATPase complex.

The yeast vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral membrane subunits involved in ATP hydrolysis and integral membrane subunits involved in proton pumping. The yeast vma21 mutant was isolated from a screen to identify mutants defective in V-ATPase function. vma21 mutants fail to assemble the V-ATPase complex onto the vacuolar membrane: peripheral subunits accumulate in the cytosol and the 100-kDa integral membrane subunit is rapidly degraded. The product of the VMA21 gene (Vma21p) is an 8.5-kDa integral membrane protein that is not a subunit of the purified V-ATPase complex but instead resides in the endoplasmic reticulum. Vma21p contains a dilysine motif at the carboxy terminus, and mutation of these lysine residues abolishes retention in the endoplasmic reticulum and results in delivery of Vma21p to the vacuole, the default compartment for yeast membrane proteins. Our findings suggest that Vma21p is required for assembly of the integral membrane sector of the V-ATPase in the endoplasmic reticulum and that the unassembled 100-kDa integral membrane subunit present in delta vma21 cells is rapidly degraded by nonvacuolar proteases.     

Structure and Assembly of the Yeast V-ATPase

The yeast V-ATPase belongs to a family of V-type ATPases present in all eucaryotic organisms. In Saccharomyces cerevisiae the V-ATPase is localized to the membrane of the vacuole as well as the Golgi complex and endosomes. The V-ATPase brings about the acidification of these organelles by the transport of protons coupled to the hydrolysis of ATP. In yeast, the V-ATPase is composed of 13 subunits consisting of a catalytic V₁ domain of peripherally associated proteins and a proton-translocating V₀ domain of integral membrane proteins. The regulatory subunit, Vma13p, was the first V-ATPase subunit to have its crystal structure determined. In addition to proteins forming the functional V-ATPase complex, three ER-localized proteins facilitate the assembly of the V₀ subunits following their translation and insertion into the membrane of the ER. Homologues of the Vma21p assembly factor have been identified in many higher eukaryotes supporting a ubiquitous assembly pathway for this important enzyme complex.     

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.     

Role of Vma21p in assembly and transport of the yeast vacuolar ATPase

The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V1) responsible for ATP hydrolysis and an integral membrane sector (V0) required for proton translocation. Biogenesis of V0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Delta cells, the major proteolipid subunit of V0 failed to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V0 subunits. Analysis of vmaDelta strains showed that binding of V0 subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V0 subunits, indicating that assembly of V0 occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V0 was transient and that Vma21p/V0 dissociation was concomitant with V0/V1 assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V0 and abrogated assembly of V0/V1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V0 in the vacuole, this interaction did not affect the assembly of vacuolar V0/V1 complexes. We conclude that Vma21p is not involved in regulating the interaction between V0 and V1 sectors, but that it has a crucial role in coordinating the assembly of V0 subunits and in escorting the assembled V0 complex into ER-derived transport vesicles.     

Evidence that the NH2 terminus of vph1p, an integral subunit of the V0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector

The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex. V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation. Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1). Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1)-sensitive ATP hydrolysis. Trypsin cleaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation.     

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.     

The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis

The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxyl-terminal domain of Stv1p. Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.     

Vma9p (subunit e) is an integral membrane V0 subunit of the yeast V-ATPase

The Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of 14 subunits distributed between a peripheral V1 subcomplex and an integral membrane V0 subcomplex. Genome-wide screens have led to the identification of the newest yeast V-ATPase subunit, Vma9p. Vma9p (subunit e) is a small hydrophobic protein that is conserved from fungi to animals. We demonstrate that disruption of yeast VMA9 results in the failure of V1 and V0 V-ATPase subunits to assemble onto the vacuole and in decreased levels of the subunit a isoforms Vph1p and Stv1p. We also show that Vma9p is an integral membrane protein, synthesized and inserted into the endoplasmic reticulum (ER), which then localizes to the limiting membrane of the vacuole. All V0 subunits and V-ATPase assembly factors are required for Vma9p to efficiently exit the ER. In the ER, Vma9p and the V0 subunits interact with the V-ATPase assembly factor Vma21p. Interestingly, the association of Vma9p with the V0-Vma21p assembly complex is disrupted with the loss of any single V0 subunit. Similarly, Vma9p is required for V0 subunits Vph1p and Vma6p to associate with the V0-Vma21p complex. In contrast, the proteolipids associate with Vma21p even in the absence of Vma9p. These results demonstrate that Vma9p is an integral membrane subunit of the yeast V-ATPase V0 subcomplex and suggest a model for the arrangement of polypeptides within the V0 subcomplex.     

Mouse proton pump ATPase C subunit isoforms (C2-a and C2-b) specifically expressed in kidney and lung

The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lung-specific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal alpha and beta intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.     

Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum

The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V₁ sector catalyzes ATP hydrolysis and the V₀ sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V₀ assembly. We have discovered a fifth V₀ assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V₀ assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V₀ assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V₀–Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V₀ subunits c, c′, and c″. On assembly of the remaining three V₀ subunits (a, d, and e) into the V₀ complex, Voa1p dissociates from the now fully assembled V₀–Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V₀ assembly in the ER, but then it dissociates before exit of the V₀–Vma21p complex from the ER for transport to the Golgi compartment.     

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.     

Defined sites of interaction between subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the stator structure of the vacuolar H+-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.     

Saccharomyces cerevisiae lacking Btn1p modulate vacuolar ATPase activity to regulate pH imbalance in the vacuole

The vacuolar H(+)-ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild-type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V(0) and V(1) V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-Delta), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1-Delta is altered. We show that down-regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1-Delta at early growth phases. We therefore propose that Btn1p is required for tight regulation of vacuolar pH to maintain the vacuolar luminal content and optimal activity of this organelle and that disruption in Btn1p function leads to a modulation of V-ATPase activity to maintain cellular pH homeostasis and vacuolar luminal content.     

Yeast V-ATPase complexes containing different isoforms of the 100-kDa a-subunit differ in coupling efficiency and in vivo dissociation

The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.     

Molecular characterization of the yeast vacuolar H+-ATPase proton pore

The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.     

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.     

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.