N,N'-dicyclohexylcarbodiimide-binding proteolipid of the vacuolar H+-ATPase from oat roots

The inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was used to probe the structure and function of the vacuolar H+-translocating ATPase from oat roots (Avena sativa var. Lang). The second-order rate constant for DCCD inhibition was inversely related to the concentration of membrane, indicating that DCCD reached the inhibitory site by concentrating in the hydrophobic environment. [14C]DCCD preferentially labeled a 16-kDa polypeptide of tonoplast vesicles, and the amount of [14C]DCCD bound to the 16-kDa peptide was directly proportional to inhibition of ATPase activity. A 16-kDa polypeptide had previously been shown to be part of the purified tonoplast ATPase. As predicted from the observed noncooperative inhibition, binding studies showed that 1 mol of DCCD was bound per mol of ATPase when the enzyme was completely inactivated. The DCCD-binding 16-kDa polypeptide was purified 12-fold by chloroform/methanol extraction. This protein was thus classified as a proteolipid, and its identity as part of the ATPase was confirmed by positive reaction with the antibody to the purified ATPase on immunoblots. From the purification studies, we estimated that the 16-kDa subunit was present in multiple (4-8) copies/holoenzyme. The purification of the proteolipid is a first step towards testing its proposed role in H+ translocation.     

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

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

Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis.

Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior surface of the membrane sector of the enzyme, contacting the lipid phase. However, structural support for this theoretical mechanism is lacking. To address this, we have used cysteine mutagenesis to produce a molecular model of the 16-kDa proteolipid complex. Transmembrane helical contacts were determined using oxidative cysteine cross-linking, and accessibility of cysteines to the lipid phase was determined by their reactivity to the lipid-soluble probe N-(1-pyrenyl)maleimide. A single model for organization of the four helices of each monomeric proteolipid was the best fit to the experimental data, with helix 1 lining a central pore and helix 2 and helix 3 immediately external to it and forming the principal intermolecular contacts. Helix 4, containing the crucial acidic residue, is peripheral to the complex. The model is consistent not only with theoretical proton transport mechanisms, but has structural similarity to the dodecameric ring complex formed by the related 8-kDa proteolipid of the F(1)F(0)-ATPase. This suggests some commonality between the proton translocating mechanisms of the vacuolar and F(1)F(0)-ATPases.     

The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site

Vacuolar H(+)-ATPase (V-ATPase) binds actin filaments with high affinity (K(d) = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164-29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin with K(d) values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.     

Ancient origin of the vacuolar H+-ATPase 69-kilodalton catalytic subunit superfamily

Recently, two distinct cDNA clones encoding the catalytic subunit of the vacuolar H⁺-ATPase (V-ATPase) were isolated from the allotetraploid cotton species Gossypium hirsutum L. cv Acala SJ-2 (Wilkins 1992, 1993). Differences in the nucleotide sequence of these clones were used as molecular markers to explore the organization and structure of the V-ATPase catalytic subunit genes in the A and D genomes of diploid and allotetraploid cotton species. Nucleotide sequencing of polymerase chain reaction (PCR) products amplified from G. arboreum (A₂, 2n=26), G. raimondii (D₅, 2n=26), and G. hirsutum cv Acala SJ-2 [(AD)₁, 2n=4x=52] revealed a V-ATPase catalytic subunit organization more complex than indicated hitherto in any species, including higher plants. In the genus Gossypium, the V-ATPase catalytic subunit genes are organized as a superfamily comprising two diverse but closely related multigene families, designated as vat69A and vat69B, present in both diploid and allotetraploid species. As expected, each vat69 subfamily is correspondingly more complex in the allotetraploid species due to the presence of both A and D alloalleles. Because of this, about one-half of the complex organization of V-ATPase catalytic subunit genes predates polyploidization and speciation of New World tetraploid species. Comparison of plant and fungal V-ATPase catalytic subunit gene structure indicates that introns accrued in the plant homologs following the bifurcation of plant and fungi but prior to the gene duplication event that gave rise to the vat69A and vat69B genes approximately 45 million years ago. The structural complexity of plant V-ATPase catalytic subunit genes is highly conserved, indicating the presence of at least ten introns dispersed throughout the coding region.     

Targeted disruption of the gene encoding the proteolipid subunit of mouse vacuolar H(+)-ATPase leads to early embryonic lethality.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for acidification of intracellular compartments in eukaryotic cells. Its 16-kDa subunit (proteolipid, PL16) plays a central role in V-ATPase function, forming the principal channel via which protons are translocated. To elucidate physiological roles of V-ATPase in mammalian cell function and embryogenesis, we attempted to generate a PL16 null mutant mouse by gene-targeting. Mice heterozygous (PL16(+/-)) for the proteolipid mutation were intercrossed and their offspring were classified according to genotype. There were no homozygous (PL16(-/-)) pups among 69 neonates examined, but a few PL16(-/-) embryos were found during the pre-implantation stages of embryonic development, up to day 3.5 post-coitum. These results suggested that PL16 (and hence V-ATPase) may play an essential role in cell proliferation and viability during early embryogenesis. PL16(+/-) mice were indistinguishable from their wild-type littermates and displayed no discernible abnormalities, although the PL16 mRNA level in PL16(+/-) mice decreased to about one-half of wild-type levels.     

Suppression of tumor-related glycosylation of cell surface receptors by the 16-kDa membrane subunit of vacuolar H+-ATPase

The glycosylation of integrins and other cell surface receptors is altered in many transformed cells. Notably, an increase in the number of beta1,6-branched N-linked oligosaccharides correlates strongly with invasive growth of cells. An ectopic expression of the Golgi enzyme N-acetylglucosaminyltransferase V (GlcNAc-TV), which forms beta1,6 linkages, promotes metastasis of a number of cell types. It is shown here that the 16-kDa transmembrane subunit (16K) of vacuolar H(+)-ATPase suppresses beta1,6 branching of beta(1) integrin and the epidermal growth factor receptor. Overexpression of 16K inhibits cell adhesion and invasion. 16K contains four hydrophobic membrane-spanning alpha-helices, and its ability to influence glycosylation is localized primarily within the second and fourth membrane-spanning alpha-helices. 16K also interacts directly with the transmembrane domain of beta(1) integrin, but its effects on glycosylation were independent of its binding to beta(1) integrin. These data link cell surface tumor-related glycosylation to a component of the enzyme responsible for acidification of the exocytic pathway.     

Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants

The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.     

Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H(+)-ATPase from a higher plant.

To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.     

A conserved gene encoding the 57-kDa subunit of the yeast vacuolar H+-ATPase

The peripheral (catalytic) sector of vacuolar H+-ATPases contains five different polypeptides denoted as subunits A-E in order of decreasing molecular masses from 72 to 33 kDa. The gene encoding subunit B (57 kDa) of yeast vacuolar H+-ATPase was cloned on a 5-kilobase pair genomic DNA fragment and sequenced. Four open reading frames were identified in the sequenced DNA. One of them encodes a protein of 504 amino acids with a calculated Mr of 56,557. Hydropathy plot revealed no apparent transmembrane segments. Southern analysis demonstrated that a single gene encodes this polypeptide in the yeast genome. The amino acid sequence exhibits extensive identity with the homologous protein from the plant Arabidopsis (77%). This polypeptide also contains regions of homology with the alpha subunits of H+-ATPases from mitochondria, chloroplasts, and bacteria. However, less similarity was detected when it was compared with the beta subunits of those enzymes. The implication of these phenomena on the evolution of proton pumps is discussed.     

H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H+-ATPase were characterized. GTP (and also ITP) was found to drive H+-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H+-ATPase, inhibited the activity at 2.5 nM. The H+-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H+-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H+-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H+-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H+-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H+-ATPase shows typical features of vacuolar H+-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.     

Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 in Xenopus pituitary.

Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a approximately 40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a approximately 27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximately 60 kDa; the nonglycosylated, newly synthesized form is approximately 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated approximately 60-kDa precursor that is intracellularly cleaved to an approximately 40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.     

On the subunit composition of the Neurospora plasma membrane H+-ATPase

The resolution-reconstitution approach has been employed in order to gain information as to the subunit composition of the Neurospora plasma membrane H+-ATPase. Proteoliposomes prepared from sonicated asolectin and a highly purified, radiolabeled preparation of the 105,000-dalton hydrolytic moiety of the H+-ATPase by a freeze-thaw procedure catalyze ATP hydrolysis-dependent proton translocation as indicated by the extensive 9-amino-6-chloro-2-methoxyacridine fluorescence quenching that occurs upon the addition of MgATP to the proteoliposomes, and the reversal of this quenching induced by the H+-ATPase inhibitor, vanadate, and the proton conductors, carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. ATP hydrolysis is tightly coupled to proton translocation into the liposomes as indicated by the marked stimulation of ATP hydrolysis by carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. The maximum stimulation of ATPase activity by proton conductors is about 3-fold, which indicates that at least two-thirds of the hydrolytically active ATPase molecules present in the reconstituted preparation are capable of translocating protons into the liposomes. Furthermore, as estimated by the extent of protection of the reconstituted 105,000-dalton hydrolytic moiety against tryptic degradation by vanadate in the presence of Mg2+ and ATP, the fraction of the total population of ATPase molecules that are hydrolytically active is at least 91%. Taken together, these data indicate that at least 61% of the ATPase molecules present in the reconstituted preparation are able to catalyze proton translocation. This information allows an estimation of the amount of any polypeptide in the preparation that must be present in order for that polypeptide to qualify as a subunit that is required for proton translocation in addition to the 105,000-dalton hydrolytic moiety, and an analysis of the radiolabeled ATPase preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea rules out the involvement of any such polypeptides larger than 2,500 daltons. This indicates that the Neurospora plasma membrane H+-ATPase has no subunits even vaguely resembling any that have been found to be associated with other transport ATPases and that if this enzyme has any subunits at all other than the 105,000-dalton hydrolytic moiety, they must be very small.     

The 16 kDa subunit of vacuolar H+-ATPase is a novel sarcoglycan-interacting protein

The sarcoglycan complex in muscle consists of alpha-, beta-, gamma- and delta-sarcoglycan and is part of the larger dystrophin-glycoprotein complex (DGC), which is essential for maintaining muscle membrane integrity. Mutations in any of the four sarcoglycans cause limb-girdle muscular dystrophies (LGMD). In this report, we have identified a novel interaction between delta-sarcoglycan and the 16 kDa subunit c (16K) of vacuolar H(+)-ATPase. Co-expression studies in heterologous cell system revealed that 16K interacts specifically with delta-sarcoglycan and the highly related gamma-sarcoglycan through the transmembrane domains. In cultured C2C12 myotubes, 16K forms a complex with sarcoglycans at the plasma membrane. Loss of sarcoglycans in the sarcoglycan-deficient BIO14.6 hamster destabilizes the DGC and alters the localization of 16K at the sarcolemma. In addition, the steady state level of beta(1)-integrin is increased. Recent studies have shown that 16K also interacts directly with beta(1)-integrin and our data demonstrated that sarcoglycans, 16K and beta(1)-integrin were immunoprecipitated together in C2C12 myotubes. Since sarcoglycans have been proposed to participate in bi-directional signaling with integrins, our findings suggest that 16K might mediate the communication between sarcoglycans and integrins and play an important role in the pathogenesis of muscular dystrophy.     

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.     

Cysteine-directed cross-linking to subunit B suggests that subunit E forms part of the peripheral stalk of the vacuolar H+-ATPase.

We have employed a combination of site-directed mutagenesis and covalent cross-linking to identify subunits in close proximity to subunit B in the vacuolar H(+)-ATPase (V-ATPase) complex. Unique cysteine residues were introduced into a Cys-less form of subunit B, and the V-ATPase complex in isolated vacuolar membranes from each mutant strain was reacted with the bifunctional, photoactivable maleimide reagent 4-(N-maleimido)benzophenone. Photoactivation resulted in cross-linking of the unique sulfhydryl groups on subunit B with other subunits in the complex. Four of the eight mutants constructed containing a unique cysteine residue at Ala(15), Lys(45), Glu(494), or Thr(501) resulted in the formation of cross-linked products, which were recognized by Western blot analysis using antibodies against both subunits B and E. These products had a molecular mass of 84 kDa, consistent with a cross-linked product of subunits B and E. Molecular modeling of subunit B places Ala(15) and Lys(45) near the top of the V(1) structure (i.e. farthest from the membrane), whereas Glu(494) and Thr(501) are predicted to reside near the bottom of V(1), with all four residues predicted to be oriented toward the external surface of the complex. A model incorporating these and previous data is presented in which subunit E exists in an extended conformation on the outer surface of the A(3)B(3) hexamer that forms the core of the V(1) domain. This location for subunit E suggests that this subunit forms part of the peripheral stalk of the V-ATPase that links the V(1) and V(0) domains.     

Topology,glycosylation and conformational changes in the membrane domain of the vacuolar H+‐ATPase a subunit

Published topological models of the integral membrane a subunit of the vacuolar proton‐translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino‐terminus and carboxyl‐terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V‐ATPase substrate, MgATP, and the V‐ATPase‐specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V‐ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle. J. Cell. Biochem. 114: 1474–1487, 2013. © 2013 Wiley Periodicals, Inc.     

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

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

A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator

Abstract

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H⁺-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal α-helical segments of the G and E subunits are closely aligned over a distance of up to 40 Å. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two ～ 150 Å long parallel α-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.