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.     

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.     

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.     

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.     

Glu-44 in the Amino-terminal α-Helix of Yeast Vacuolar ATPase E Subunit (Vma4p) Has a Role for VoV1 Assembly

The proton (H⁺) pumping vacuolar-type ATPase (V-ATPase) is a rotary enzyme that plays a pivotal role in forming intracellular acidic compartments in eukaryotic cells. In Saccharomyces cerevisiae, the membrane extrinsic catalytic V₁ and the transmembrane proton-pumping V_o complexes have been shown to reversibly dissociate upon removal of glucose from the medium. However, the basis of this disassembly is largely unknown. In the earlier study, we have found that the amino-terminal α-helical domain between Lys-33 and Lys-83 of yeast E subunit (Vma4p) in the peripheral stalk of the V₁ complex has a role in glucose-dependent V_oV₁ assembly. Results of alanine-scanning mutagenesis within the domain revealed that the Vma4p Glu-44 is a key residue in V_oV₁ disassembly. Biochemical analysis on Vma4p Glu-44 to Ala, Asn, Asp, and Gln substitutions indicated that Glu-44 has a role in V-ATPase catalysis. These results suggest that Glu-44 is one of the key functional residues for subunit interaction in the V-ATPase stalk complex that allows both efficient rotation catalysis and assembly.     

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.     

Assembly of the Yeast Vacuolar Proton-Translocating ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is the bestcharacterized member of the V-ATPase family. Biochemical and genetic screensled to the identification of a large number of genes in yeast, designatedVMA, encoding proteins required to assemble a functional V-ATPase. Atotal of thirteen genes encode subunits of the final enzyme complex. Inaddition to subunit-encoding genes, we have identified three genes that codefor proteins that are not part of the final V-ATPase complex yet required forits assembly. We refer to these nonsubunit Vma proteins as assembly factors,since their function is dedicated to assembling the V-ATPase. The assemblyfactors, Vma12p, Vma21p, and Vma22p are localized to the endoplasmicreticulum (ER) and aid the assembly of newly synthesized V-ATPase subunitsthat are translocated into the ER membrane. At least two of these proteins,Vma12p and Vma22p, function together in an assembly complex and interactdirectly with nascent V-ATPase subunits.     

Characterization of the complex involved in regulating V-ATPase activity of the vacuolar and endosomal membrane

Regulator of the H⁺-ATPase of the vacuolar and endosomal membranes (RAVE) is essential for the reversible assembly of H⁺-ATPase. RAVE primarily consists of three subunits: Rav1p, Rav2p and Skp1p. To characterize these subunits, in this study, four strains derived from Saccharomyces cerevisiae BY4742 were constructed with a FLAG tag on the Rav1p and Rav2p subunits. Then, the corresponding RAVE containing complex was isolated by affinity purification. Western blot and MALDI-TOF mass spectrometry analyses showed that the RAVE complex contains not only the known V₁-ATPase subunits (Vma1p and Vma2p) but also a newly found Leu1p that interacts with the RAVE subunit. Furthermore, we constructed rav1?/rav2?/vma2?/leu1-deficient recombinants by fusion PCR and homologous recombination and demonstrated that leu1 is indispensable in adjusting the microbial cell to adverse environments and that the function is similar to that of rav1/rav2 but significantly differs from that of vma2. Leu1p probably plays an important role in RAVE regulation of V-ATPase activity in conjunction with RAVE.     

Interaction between subunit C (Vma5p) of the yeast vacuolar ATPase and the stalk of the C-depleted V1 ATPase from Manduca sexta midgut

Projection maps of a V₁-Vma5p hybrid complex, composed of subunit C (Vma5p) of Saccharomyces cerevisiae V-ATPase and the C-depleted V₁ from Manduca sexta, were determined from single particle electron microscopy. V₁-Vma5p consists of a headpiece and an elongated wedgelike stalk with a 2.1×3.0 nm protuberance and a 9.5×7.5 globular domain, interpreted to include Vma5p. The interaction face of Vma5p in V₁ was explored by chemical modification experiments.     

A genome-wide enhancer screen implicates sphingolipid composition in vacuolar ATPase function in Saccharomyces cerevisiae

The function of the vacuolar H(+)-ATPase (V-ATPase) enzyme complex is to acidify organelles; this process is critical for a variety of cellular processes and has implications in human disease. There are five accessory proteins that assist in assembly of the membrane portion of the complex, the V(0) domain. To identify additional elements that affect V-ATPase assembly, trafficking, or enzyme activity, we performed a genome-wide enhancer screen in the budding yeast Saccharomyces cerevisiae with two mutant assembly factor alleles, VMA21 with a dysfunctional ER retrieval motif (vma21QQ) and vma21QQ in combination with voa1Δ, a nonessential assembly factor. These alleles serve as sensitized genetic backgrounds that have reduced V-ATPase enzyme activity. Genes were identified from a variety of cellular pathways including a large number of trafficking-related components; we characterized two redundant gene pairs, HPH1/HPH2 and ORM1/ORM2. Both sets demonstrated synthetic growth defects in combination with the vma21QQ allele. A loss of either the HPH or ORM gene pairs alone did not result in a decrease in vacuolar acidification or defects in V-ATPase assembly. While the Hph proteins are not required for V-ATPase function, Orm1p and Orm2p are required for full V-ATPase enzyme function. Consistent with the documented role of the Orm proteins in sphingolipid regulation, we have found that inhibition of sphingolipid synthesis alleviates Orm-related growth defects.     

Degradation of the gluconeogenic enzyme fructose-1, 6-bisphosphatase is dependent on the vacuolar ATPase

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to Vid (vacuolar import and degradation) vesicles and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar- H+ -ATPase (V-ATPase) have been identified repeatedly. The V-ATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that mutants lacking Stv1p, Vph1p, and other subunits of the V-ATPase are defective for FBPase degradation. FBPase was detected in Vid vesicles. However, most FBPase was resistant to proteinase K digestion in the Deltavph1 or vma mutants, whereas the majority of FBPase was sensitive to proteinase K digestion in the Deltastv1 mutant. Therefore, STV1 and VPH1 have distinct functions in FBPase degradation. In cells lacking V0 genes, Vma2p and Vma5p were still detected on Vid vesicles and vacuoles, suggesting that the distribution of V1 proteins is independent of V0 genes. The V0 and V1 domains are assembled following a glucose shift and the assembly is not regulated by protein kinase A and RAV genes. Assembly of the V0 complex is necessary for FBPase trafficking, since mutants that block the assembly and transport of V0 out of the ER were defective in FBPase degradation.     

Structural bases of physiological functions and roles of the vacuolar H(+)-ATPase

Vacuolar-type H⁺-ATPases (V-ATPases) is a large multi-protein complex containing at least 14 different subunits, in which subunits A, B, C, D, E, F, G, and H compose the peripheral 500-kDa V₁ responsible for ATP hydrolysis, and subunits a, c, c′, c″, and d assembly the 250-kDa membrane-integral V₀ harboring the rotary mechanism to transport protons across the membrane. The assembly of V-ATPases requires the presence of all V₁ and V₀ subunits, in which the V₁ must be completely assembled prior to association with the V₀, accordingly the V₀ failing to assemble cannot provide a membrane anchor for the V₁, thereby prohibiting membrane association of the V-ATPase subunits. The V-ATPase mediates acidification of intracellular compartments and regulates diverse critical physiological processes of cell for functions of its numerous functional subunits. The core catalytic mechanism of the V-ATPase is a rotational catalytic mechanism. The V-ATPase holoenzyme activity is regulated by the reversible assembly/disassembly of the V₁ and V₀, the targeting and recycling of V-ATPase-containing vesicles to and from the plasma membrane, the coupling ratio between ATP hydrolysis and proton pumping, ATP, Ca²⁺, and its inhibitors and activators.     

Regulation of yeast ectoapyrase ynd1p activity by activator subunit Vma13p of vacuolar H+-ATPase

CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. By using a two-hybrid screen, we found that an activator subunit (Vma13p) of yeast vacuolar H(+)-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation. Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Delta mutant was drastically increased compared with that of Ynd1p in VMA13 cells. A similar increase in the apyrase activity of Ynd1p was found in a vma1Delta mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes. However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels of Ynd1p activity, demonstrating that the presence of Vma13p rather than the function of V-ATPase in the membrane represses Ynd1p activity. These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.     

Reversible Association between the V1 and V0 Domains of Yeast Vacuolar H+-ATPase Is an Unconventional Glucose-Induced Effect

The yeast vacuolar H⁺-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V₁), containing the ATP binding sites, and an integral membrane domain (V₀), forming the proton pore. Dissociation of the V₁ and V₀ domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V₁ and V₀ domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.     

Arabidopsis has two functional orthologs of the yeast V-ATPase assembly factor Vma21p

How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

Mutations in ATP6AP2 cause autophagic liver disease in humans

The biogenesis of the proton pump V-ATPase commences with the assembly of the proton pore sector V₀ in the endoplasmic reticulum (ER). This process occurs under the control of a group of assembly factors whose mutations have recently been shown to cause glycosylation disorders with overlapping phenotypes in humans. Using whole exome sequencing, we demonstrate that mutations of the accessory V-ATPase subunit ATP6AP2 cause a similar disease characterized by hepatosteatosis, lipid abnormalities, immunodeficiency and cognitive impairment. ATP6AP2 interacts with members of the V₀ assembly complex, and its ER localization is crucial for V-ATPase activity. Moreover, ATP6AP2 mutations can cause developmental defects and steatotic phenotypes when introduced into Drosophila. Altogether, our data suggest that these phenotypes are the result of a pathogenetic cascade that includes impaired V-ATPase assembly, defective lysosomal acidification, reduced MTOR signaling and autophagic misregulation.     

The signaling lipid PI(3,5)P2 stabilizes V1–Vo sector interactions and activates the V-ATPase

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V₁ domain from the integral membrane V_o domain. Multiple stresses induce changes in V₁-V_o assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂). V-ATPase activation through V₁-V_o assembly in response to salt stress is strongly dependent on PI(3,5)P₂ synthesis. Purified V_o complexes preferentially bind to PI(3,5)P₂ on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P₂ levels in vivo recruits the N-terminal domain of V_o-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V₁-V_o interaction, suggesting that interaction of Vph1p with PI(3,5)P₂-containing membranes stabilizes V₁-V_o assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1∆ and vac14∆ mutants and suggest that human disease phenotypes associated with PI(3,5)P₂ loss may arise from compromised V-ATPase stability and regulation.     

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.