The E and G subunits of the yeast V-ATPase interact tightly and are both present at more than one copy per V1 complex

The E and G subunits of the yeast V-ATPase are believed to be part of the peripheral or stator stalk(s) responsible for physically and functionally linking the peripheral V1 sector, responsible for ATP hydrolysis, to the membrane V0 sector, containing the proton pore. The E and G subunits interact tightly and specifically, both on a far Western blot of yeast vacuolar proteins and in the yeast two-hybrid assay. Amino acids 13-79 of the E subunit are critical for the E-G two-hybrid interaction. Different tagged versions of the G subunit were expressed in a diploid cell, and affinity purification of cytosolic V1 sectors via a FLAG-tagged G subunit resulted in copurification of a Myc-tagged G subunit, implying more than one G subunit was present in each V1 complex. Similarly, hemagglutinin-tagged E subunit was able to affinity-purify V1 sectors containing an untagged version of the E subunit from heterozygous diploid cells, suggesting that more than one E subunit is present. Overexpression of the subunit G results in a destabilization of subunit E similar to that seen in the complete absence of subunit G (Tomashek, J. J., Graham, L. A., Hutchins, M. U., Stevens, T. H., and Klionsky, D. J. (1997) J. Biol. Chem. 272, 26787-26793). These results are consistent with recent models showing at least two peripheral stalks connecting the V1 and V0 sectors of the V-ATPase and would allow both stalks to be based on an EG dimer.     

Peripheral stator of the yeast V-ATPase: stoichiometry and specificity of interaction between the EG complex and subunits C and H

V-ATPases are multisubunit membrane protein complexes that use the energy provided by ATP hydrolysis to generate a proton gradient across various intracellular and plasma membranes. In doing so, they maintain an acidic pH in the lumen of intracellular organelles and acidify extracellular milieu to support specific cellular functions. V-ATPases are structurally similar to the F1F0-ATP synthase, with an intrinsic membrane domain (V0) and an extrinsic peripheral domain (V1) joined by several connecting elements. To gain a clear functional understanding of the catalytic mechanism, and of the stability requirements for regulatory processes in the enzyme, a clear topology of the enzyme has to be established. In particular, the composition and arrangement of the peripheral stator subunits must be firmly settled, as these play specific roles in catalysis and regulation. We have designed a strategy allowing us to coexpress different combinations of these subunits to delineate specific interactions. In this study, we report the interaction between the peripheral stator EG complex and subunits C and H of the V-ATPase from the yeast Saccharomyces cerevisae. A combination of analytical gel filtration, native gel electrophoresis, and ultracentrifugation analysis allowed us to ascertain the homogeneity and molar mass of the purified EGC complex as well as of the EG complex, supporting the formation of 1:1(:1) stoichiometric complexes. The EGC complex can be formed in vitro by combining equimolar amounts of subunit C and the EG subcomplex and results most likely from the initial interaction between subunits E and C.     

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.     

Structure of the yeast vacuolar ATPase

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.     

Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

RAVE is essential for the efficient assembly of the C subunit with the vacuolar H(+)-ATPase

The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V(1) and V(0) sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V(1) peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V(1) complexes, suggesting that RAVE may play a role in docking of the V(1) peripheral stalk to the V(0) complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V(1) subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V(1) and V(0) sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V(1)/V(0) complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V(1) peripheral stalk subunit interactions with V(0) during V-ATPase assembly.     

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.     

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.     

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.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Distinct expression patterns of different subunit isoforms of the V-ATPase in the rat epididymis

In the epididymis and vas deferens, the vacuolar H(+)ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.     

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 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.     

Interaction between aldolase and vacuolar H+-ATPase: evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pump

Vacuolar H(+)-ATPases (V-ATPases) are essential for acidification of intracellular compartments and for proton secretion from the plasma membrane in kidney epithelial cells and osteoclasts. The cellular proteins that regulate V-ATPases remain largely unknown. A screen for proteins that bind the V-ATPase E subunit using the yeast two-hybrid assay identified the cDNA clone coded for aldolase, an enzyme of the glycolytic pathway. The interaction between E subunit and aldolase was confirmed in vitro by precipitation assays using E subunit-glutathione S-transferase chimeric fusion proteins and metabolically labeled aldolase. Aldolase was isolated associated with intact V-ATPase from bovine kidney microsomes and osteoclast-containing mouse marrow cultures in co-immunoprecipitation studies performed using an anti-E subunit monoclonal antibody. The interaction was not affected by incubation with aldolase substrates or products. In immunocytochemical assays, aldolase was found to colocalize with V-ATPase in the renal proximal tubule. In osteoclasts, the aldolase-V-ATPase complex appeared to undergo a subcellular redistribution from perinuclear compartments to the ruffled membranes following activation of resorption. In yeast cells deficient in aldolase, the peripheral V(1) domain of V-ATPase was found to dissociate from the integral membrane V(0) domain, indicating direct coupling of glycolysis to the proton pump. The direct binding interaction between V-ATPase and aldolase may be a new mechanism for the regulation of the V-ATPase and may underlie the proximal tubule acidification defect in hereditary fructose intolerance.     

The RAVE complex is essential for stable assembly of the yeast V-ATPase

Vacuolar proton-translocating ATPases are composed of a peripheral complex, V(1), attached to an integral membrane complex, V(o). Association of the two complexes is essential for ATP-driven proton transport and is regulated post-translationally in response to glucose concentration. A new complex, RAVE, was recently isolated and implicated in glucose-dependent reassembly of V-ATPase complexes that had disassembled in response to glucose deprivation (Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384-391). Here, we provide evidence supporting a role for RAVE in reassembly of the V-ATPase but also demonstrate an essential role in V-ATPase assembly under other conditions. The RAVE complex associates reversibly with V(1) complexes released from the membrane by glucose deprivation but binds constitutively to cytosolic V(1) sectors in a mutant lacking V(o) sectors. V-ATPase complexes from cells lacking RAVE subunits show serious structural and functional defects even in glucose-grown cells or in combination with a mutation that blocks disassembly of the V-ATPase. RAVE small middle dotV(1) interactions are specifically disrupted in cells lacking V(1) subunits E or G, suggesting a direct involvement for these subunits in interaction of the two complexes. Skp1p, a RAVE subunit involved in many different signal transduction pathways, binds stably to other RAVE subunits under conditions that alter RAVE small middle dotV(1) binding; thus, Skp1p recruitment to the RAVE complex does not appear to provide a signal for V-ATPase assembly.     

Localization of subunits D,E, and G in the yeast V-ATPase complex using cysteine-mediated cross-linking to subunit B

Using a combination of cysteine mutagenesis and covalent cross-linking, we have identified subunits in close proximity to specific sites within subunit B of the vacuolar (H(+))-ATPase (V-ATPase) of yeast. Unique cysteine residues were introduced into subunit B by site-directed mutagenesis, and the resultant V-ATPase complexes were reacted with the bifunctional, photoactivatable maleimide reagent 4-(N-maleimido)benzophenone (MBP) followed by irradiation. Cross-linked products were identified by Western blot using subunit-specific antibodies. Introduction of cysteine residues at positions Glu(106) and Asp(199) led to cross-linking of subunits B and E, at positions Asp(341) and Ala(424) to cross-linking of subunits B and D, and at positions Ala(15) and Lys(45) to cross-linking of subunits B and G. Using a molecular model of subunit B constructed on the basis of sequence homology between the V- and F-ATPases, the X-ray coordinates of the F(1)-ATPase, and energy minimization, Glu(106), Asp(199), Ala(15), and Lys(45) are all predicted to be located on the outer surface of the complex, with Ala(15) and Lys(45) located near the top of the complex furthest from the membrane. By contrast, Asp(341) and Ala(424) are predicted to face the interior of the A(3)B(3) hexamer. These results suggest that subunits E and G form part of a peripheral stalk connecting the V(1) and V(0) domains whereas subunit D forms part of a central stalk. Subunit D is thus the most likely homologue to the gamma subunit of F(1), which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.     

Structural characterization of an ATPase active F1-/V1 -ATPase (alpha3beta3EG) hybrid complex

Co-reconstitution of subunits E and G of the yeast V-ATPase and the alpha and beta subunits of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) resulted in an alpha(3)beta(3)EG hybrid complex showing 53% of the ATPase activity of TF(1). The alpha(3)beta(3)EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2-2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the alpha(3)beta(3) subcomplex from TF(1) (Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) Structure 5, 825-836) demonstrates that a seventh mass is located inside the shaft of the alpha(3)beta(3) barrel and extends out from the hexamer. Furthermore, difference mapping of the alpha(3)beta(3)EG oligomer with projections of the A(3)B(3)E and A(3)B(3)EC subcomplexes of the V(1) from Caloramator fervidus (Chaban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO Rep. 3, 982-987) shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active alpha(3)beta(3)EG hybrid complex indicates that the coupling subunit gamma inside the alpha(3)beta(3) oligomer of F(1) can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and gamma subunits are structurally similar, despite the fact that their genes do not show significant homology.     

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.     

The C-H Peripheral Stalk Base: A Novel Component in V1-ATPase Assembly

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V₁V_o-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V₁-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE₃G₃H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembly mechanism of the free V₁-ATPase.