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W2130261095 abstract "Vacuolar ATPases are ATP hydrolysis-driven proton pumps found in the endomembrane system of eucaryotic cells where they are involved in pH regulation. We have determined the three-dimensional structure of the proton channel domain of the vacuolar ATPase from bovine brain clathrin-coated vesicles by electron microscopy at 21 Å resolution. The model shows an asymmetric protein ring with two small openings on the luminal side and one large opening on the cytoplasmic side. The central hole on the luminal side is covered by a globular protein, while the cytoplasmic opening is covered by two elongated proteins arranged in a collar-like fashion. Vacuolar ATPases are ATP hydrolysis-driven proton pumps found in the endomembrane system of eucaryotic cells where they are involved in pH regulation. We have determined the three-dimensional structure of the proton channel domain of the vacuolar ATPase from bovine brain clathrin-coated vesicles by electron microscopy at 21 Å resolution. The model shows an asymmetric protein ring with two small openings on the luminal side and one large opening on the cytoplasmic side. The central hole on the luminal side is covered by a globular protein, while the cytoplasmic opening is covered by two elongated proteins arranged in a collar-like fashion. vacuolar type ATPase multivariate statistical analysis In eucaryotic cells, acidification and energization of organelles such as Golgi-derived vesicles, clathrin coated vesicles, synaptic vesicles, lysosomes, and the plant vacuole are accomplished by a vacuolar type ATPase (V-ATPase)1, and its proton pumping action plays a vital role in processes like protein trafficking, receptor-mediated endocytosis, neurotransmitter release, intracellular pH regulation, and waste management (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar, 2Finbow M.E. Harrison M.A. Biochem. J. 1997; 324: 697-712Crossref PubMed Scopus (230) Google Scholar, 3Nelson N. Harvey W.R. Physiol. Rev. 1999; 79: 361-385Crossref PubMed Scopus (367) Google Scholar). The vacuolar ATPase contains a large cytoplasmic ATPase domain (V1) and a membrane-embedded proton channel, V0. The two parts are connected via a stalk structure that functions to transmit the energy released during ATP hydrolysis taking place on the V1 to drive proton translocation across the membrane-bound V0. The eucaryotic V-ATPase is composed of 13 different polypeptides, with molecular masses ranging from 13 to 100 kDa. Eight of these, subunits A-H, form the cytoplasmic ATPase domain, while the membrane-embedded proton channel is made of the remaining five, subunits a, c, c′, c“ and d. A 14th subunit, Ac45, which is associated with the membrane-bound domain, is present in the mammalian enzyme in some tissues (4Supek F. Supekova L. Mandiyan S. Pan Y-C. Nelson H. Nelson N. J. Biol. Chem. 1994; 269: 24102-24106Abstract Full Text PDF PubMed Google Scholar).The V-ATPase is structurally and functionally related to the F1F0-ATPsynthase, and it is believed that both enzymes have evolved from a common ancestor (5Gogarten J.P. Kibak H. Dittrich P. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Date T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (531) Google Scholar). In case of the F-ATPase, a high resolution crystal structure exists for the cytoplasmic domain (6Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2733) Google Scholar), and based on the degree of sequence identity between the catalytic subunits of the F- and V-ATPase, it is likely that the V1-ATPase catalytic core has a similar three-dimensional-fold. The functional element proton channel is less well understood mainly because there is no detailed structure available for the intact membrane domain of either V- or F-ATPase. It is generally assumed that the c subunits are arranged in a ring-like fashion with the a subunit bound at the outside of the ring, and there is evidence for such an arrangement from electron microscopic images of both enzymes (7Wilkens S. Capaldi R.A. Nature. 1998; 393: 29Crossref PubMed Scopus (135) Google Scholar, 8Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Furthermore, it is believed that the proton channel is formed at the interface of the aand c subunits and that a rotation of the csubunit ring against the a subunit is an integral part of the mechanism of ATP hydrolysis-coupled proton translocation (9Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (432) Google Scholar).The structure of the proton channel-forming c subunit of the F-ATPase from Escherichia coli has been determined by nuclear magnetic resonance (NMR) spectroscopy (10Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 25: 8817-8824Crossref Scopus (271) Google Scholar), and it is known from low resolution x-ray (11Stock D. Leslie A.G.W. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1077) Google Scholar), atomic force microscopy, (12Seelert H. Poetsch A. Dencher N.A. Engel A. Stahlberg H. Muller D.J. Nature. 2000; 405: 418-419Crossref PubMed Scopus (413) Google Scholar) and electron microscopy studies (13Stahlberg H. Müller D.J. Suda K. Fotiadis D. Engel A. Meier T. Matthey U. Dimroth P. EMBO Rep. 2001; 2: 229-233Crossref PubMed Scopus (168) Google Scholar) that isolated F-ATPase proteolipids from different species are able to form symmetric oligomeric rings of between 20 and 28 transmembrane α helices. The same is true for isolated V-ATPase proteolipids in that they are able to form symmetric six-membered rings as seen in ordered gap junction sheets from Nephrops norvegicus, which are entirely formed by a protein identical to the V-ATPase c subunit (14Páli T. Finbow M.E. Holzenburg A. Findlay J.B. Marsh D. Biochemistry. 1995; 34: 9211-9218Crossref PubMed Scopus (35) Google Scholar).The situation in the V-ATPase membrane domain is somewhat more complex in that its c subunit ring contains three different proteolipid isoforms, namely c, c′, and c“. This means that the proteolipid ring in the V0, in contrast to its F-ATPase counterpart, is most likely asymmetric depending on whether all three proteolipid isoforms are part of the ring and what their stoichiometry is. While the ring-like arrangement of the isolated proteolipids is now well established, there is essentially no structural information available with regard to the intact, subunit a-containing proton channel. Here we report the three-dimensional structure of the V0 domain determined at a resolution of 21 Å by angular reconstitution from electron microscopic images of a negatively stained specimen. The model is discussed in the context of the conventional proton pumping function of the V0 but also with respect to a recent report that the free V0 domain may play an important role in vacuolar membrane fusion (15Peters C. Bayer M. Bühler S. Andersen J. Mann M. Mayer A. Nature. 2001; 409: 581-588Crossref PubMed Scopus (421) Google Scholar).RESULTS AND DISCUSSIONFree V-ATPase membrane domain can be found in endosomal membranes including the membranes of bovine brain clathrin-coated vesicles where it exists in excess over the intact V-ATPase complex (16Zhang J. Myers M. Forgac M. J. Biol. Chem. 1992; 267: 9773-9778Abstract Full Text PDF PubMed Google Scholar). In contrast to the F-ATPase proton channel, the free V0 complex does not function as a passive proton pore (16Zhang J. Myers M. Forgac M. J. Biol. Chem. 1992; 267: 9773-9778Abstract Full Text PDF PubMed Google Scholar, 20Schneider E. Altendorf K. EMBO J. 1985; 4: 515-518Crossref PubMed Scopus (109) Google Scholar) and it can be purified as a stable complex containing subunits a,c, c′, c“, d, and Ac45 (16Zhang J. Myers M. Forgac M. J. Biol. Chem. 1992; 267: 9773-9778Abstract Full Text PDF PubMed Google Scholar). Fig. 1 A shows SDS-polyacrylamide gel electrophoresis of coated vesicle V0domain used in this study. Glycoprotein Ac45 tends to run as a rather diffused band on polyacrylamide gels (4Supek F. Supekova L. Mandiyan S. Pan Y-C. Nelson H. Nelson N. J. Biol. Chem. 1994; 269: 24102-24106Abstract Full Text PDF PubMed Google Scholar) and is best visualized in the gel by prolonged development of the silver stain (right lanein Fig. 1 A). Fig. 1 B gives a graphic representation of the subunits contained in the coated vesicle V0. The a subunit is a two-domain protein with a hydrophilic N-terminal and a membrane-bound C-terminal domain. Studies conducted with the yeast enzyme indicate that the C terminus contains nine transmembrane α helices while the N-terminal half forms a domain exposed to the cytoplasm (21Leng X.H. Nishi T. Forgac M. J. Biol. Chem. 1999; 274: 14655-14661Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The c and c′ subunits contain four predicted membrane-spanning α helices, and it is assumed that the corresponding gene has evolved through a fusion of an ancestral two-transmembrane α helix proteolipid preserved in the F-ATPase (5Gogarten J.P. Kibak H. Dittrich P. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Date T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (531) Google Scholar). The larger c” isoform displays about 30% sequence identity to the c isoform and contains five predicted transmembrane α helices. All three isoforms contain lipid-exposed glutamate residues (c“ contains two, but only one is essential; (22Umemoto N. Ohya Y. Anraku Y. J. Biol. Chem. 1991; 266: 24526-245232Abstract Full Text PDF PubMed Google Scholar)) and these glutamates together with polar residues in the C-terminal domain of the a subunit are essential for proton pumping (22Umemoto N. Ohya Y. Anraku Y. J. Biol. Chem. 1991; 266: 24526-245232Abstract Full Text PDF PubMed Google Scholar, 23Leng X.H. Manolson M.F. Liu Q. Forgac M. J. Biol. Chem. 1996; 271: 22487-22493Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), suggesting that all three isoforms are part of the proteolipid ring. Subunit d is not membrane-anchored and may be connected to the V0 by interaction with the a subunit cytoplasmic domain (24Graham L.A. Powell B. Stevens T.H. J. Exp. Biol. 2000; 203: 61-70Crossref PubMed Google Scholar). Glycoprotein Ac45 is predicted to be oriented toward the luminal side of the complex anchored to the membrane by a single α helix in the C terminus of the polypeptide (4Supek F. Supekova L. Mandiyan S. Pan Y-C. Nelson H. Nelson N. J. Biol. Chem. 1994; 269: 24102-24106Abstract Full Text PDF PubMed Google Scholar).The stoichiometry of the V0 subunits has been determined by quantitative amino acid analysis for the coated vesicle V0to a1(c,c′)5–6 c“1 and d1, respectively, assuming that the polypeptide migrating with an apparent molecular mass of 19,000 is subunit c” (25Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar, 26Xu T. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 28909-28915Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Consistent with this assumption are studies in yeast that indicate that both the c′ and c“ isoforms are present in each one copy per complex (27Powell B. Graham L.A. Stevens T.H. J. Biol. Chem. 2000; 275: 23654-23660Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Ac45 was found to be present in varying amounts (26Xu T. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 28909-28915Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), possibly a result of the fact that clathrin-coated vesicles are derived from both the plasma membrane and the Golgi and that Ac45 might be present only in one of these membranes.Fig. 2 summarizes electron microscopy and image analysis of the V0. Fig. 2 Ashows electron microscopy of the negatively stained V0domain. In the images, the complex appears as a rather featureless globular object. Close inspection of the projections showed that the majority of the molecules had the appearance of a small disc with small globular densities on each side while others seemed to be more or less circular with no discernible features (see arrows in Fig.2 A). A data-set of 10,259 V0 images was collected and subjected to single particle image analysis with the IMAGIC 5 package of programs (17van Heel M. Harauz G. Orlova E.V Schmidt R. Schatz M. J. Struct. Biol. 1996; 116: 17-24Crossref PubMed Scopus (1044) Google Scholar). Fig. 2 B shows two averages that were obtained by the bias-free “alignment by classification” protocol (28Dube P. Tavares P. Lurz R. van Heel M. EMBO J. 1993; 12: 1303-1309Crossref PubMed Scopus (226) Google Scholar). Members of the class whose average is shown in image 2 of Fig. 2 B are oriented on the carbon film to produce the so-called side-view projection in which the complex is seen parallel to the membrane bilayer. This projection of the V0 corresponds to the projection of the V0 in the intact V-ATPase (see Fig. 2 C; taken from Ref. 8Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), allowing assignment of the cytoplasmic and luminal side of the V0 complex. The other projections are from complexes seen more or less normal to the bilayer (image 1 in Fig. 2 B). The data set was then treated by several rounds of multireference alignment and MSA/classification to extract all the characteristic views of the complex, some of which are shown in Fig. 2 D.Figure 2Electron microscopy and image analysis of the V0 domain. A, electron microscopy of purified V0 domain, negatively stained with 1% uranyl acetate. Electron optical magnification ×47,000. B,two averages obtained by the alignment by classification procedure showing a top view (image 1) and a side view (image 2) of the V0 domain. C, intact V-ATPase (taken from Ref.8Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) showing the orientation of the complex in the bilayer. D,subset of averages obtained after several rounds of multireference alignment and MSA/classification.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A visual inspection of the projections shown in Fig. 2 Dsuggested that their variation in viewing directions might be sufficient for calculating a three-dimensional model of the V-ATPase membrane domain. A set of 33 projection images, including the ones shown in Fig. 2 D, was used for starting up the angular reconstitution procedure as implemented in IMAGIC 5. The structure was refined with an increasing number of input projections leading to a model that was calculated from 97 class averages. The model was then forward-projected along 50 directions distributed uniformly over the entire Euler sphere, and the projections were used as references in a multireference alignment step leading to new input projections. The procedure was iterated until in the end 288 forward projections were used as references resulting in a total of 220 input averages from which the final three-dimensional model was calculated. Fig.3 A shows that the viewing directions are distributed fairly well over the entire Euler sphere indicating essentially isotropic information content in the structure. The resolution of the final model was estimated via the Fourier shell correlation computed between two reconstructions calculated from randomly selected subsets of the input images (Fig. 3 B). At 0.5 correlation (18Böttcher B. Wynne S.A. Crowther R.A. Nature. 1997; 386: 88-91Crossref PubMed Scopus (691) Google Scholar), the spatial frequency was 0.47 nm−1corresponding to a resolution of 21 Å, while the 3ς criterion (19Schatz M. Orlova E.V. Dube P. Jager J. van Heel M. J. Struct. Biology. 1995; 114: 28-40Crossref PubMed Scopus (111) Google Scholar) indicated that the model contains information down to 18 Å resolution.Figure 3Three-dimensional reconstruction. A, distribution of viewing angles of the class sums used for calculating the three-dimensional model of the V0. The red coordinates indicate views form the backside of the Euler sphere. B, Fourier shell correlation (●) of two models, each calculated from 110 averages randomly selected from the total of 220 input projections. The 0.5 correlation criterion indicates a resolution of ∼21 Å, whereas the 3ς criterion (♦) shows that the model contains information down to 18 Å resolution.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4 summarizes the three-dimensional structure of the V-ATPase membrane domain. Overall, the structure can be described as an asymmetric protein ring with dimensions 11 × 14 × 6 nm (including detergent and lipid), which is capped by protein densities on both sides. One side (see arrow in image 1, Fig. 4 A) is covered by a globular density with a diameter of ∼4 nm. The other side is covered by two elongated proteins, ∼7 × 3 and 6 × 3 nm, respectively, that are arranged as two half-circles (see arrows in image 2, Fig.4 A). One of these two densities is in contact with the asymmetric protein ring while the other is above the ring, connected to the first through a small bridge in the center. The orientation of the V0 in the membrane is such that the single globular density is directed toward the lumen of the vesicle (see Fig. 2 C) suggesting that this density is Ac45 for which a luminal orientation had been predicted (4Supek F. Supekova L. Mandiyan S. Pan Y-C. Nelson H. Nelson N. J. Biol. Chem. 1994; 269: 24102-24106Abstract Full Text PDF PubMed Google Scholar). The narrow connection between the luminal density and the proteolipid ring would be consistent with a single membrane anchoring α helix in the C terminus of the polypeptide (4Supek F. Supekova L. Mandiyan S. Pan Y-C. Nelson H. Nelson N. J. Biol. Chem. 1994; 269: 24102-24106Abstract Full Text PDF PubMed Google Scholar). Additional evidence that supports this assignment comes from electron microscopy of the yeast V-ATPase complex, which does not contain a homologue to Ac45 and which does not show as such a luminal density. 2S. Wilkens and M. Forgac, unpublished results. As mentioned above, Ac45 might be present in less than one copy per complex (between 0.3 and 0.7) as revealed by quantitative amino acid analysis performed with the holo enzyme (26Xu T. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 28909-28915Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The ratio might be somewhat closer to one for the free V0 found in clathrin-coated vesicles as no class-sums of the side-view projection have been obtained in which the luminal density is missing. It can, of course, not be ruled out that some of the other projections do not have the luminal density bound; the clear presence of the luminal protein in the three-dimensional reconstruction, however, suggests that this is probably not the case to a significant degree. Assignment of Ac45 leaves the N-terminal domain of subunit a and subunit d for the proteins covering the cytoplasmic opening of the channel, again consistent with their predicted orientation.Figure 4Three-dimensional structure of the V0 domain. A, surface views of the V0 domain. The volume used to represent the surface is 470 nm3 corresponding to a relative molecular mass of ∼400,000 (300,000 for the protein and an estimated 100,000 for a stain-excluding layer of detergent and tightly bound lipid). Images 2, 3, and 4 are generated from image 1 by a 90° rotation around the horizontal, a −120° rotation around the vertical, and a −90° rotation around the horizontal, respectively. The arrows in images 1 and 2 indicate the positions of the luminal density and the two cytoplasmic proteins, respectively. B, the inside of the channel as seen toward the lumen (image 1) and the cytoplasm (image 2). The two arrowheads in image 1 indicate the luminal openings of the channel. C, cross-sections of the V0 at the positions indicated on the left in image 1, part A). The sections are rotated 45° with respect to the surface representations. The arrow in image 5 in the bottom row indicates the position of the putative a subunit C terminus, while the two arrowheads point to a density that might be formed by one of the proteolipids. Bar = 4 nm in A and B) and 6 nm in C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4 B shows the V0 model with either the top half (image 1) or bottom half (image 2) removed to allow a view inside the channel. The bottom half, looking down toward the lumen, contains two openings roughly 2 nm in diameter, one of which is covered by the single globular protein (see arrowheads in Fig.4 B, image 1). The cytoplasmic opening, which is covered by the two elongated proteins (see arrows in Fig.4 A, image 2), has a diameter of ∼6 nm (Fig. 4 B, image 2). This would imply that the ring forming proteolipids is not arranged strictly perpendicular to the bilayer so they would form a cylinder but rather at an angle to one another to produce a funnel-shaped structure with a smaller luminal and a larger cytoplasmic opening. Channel openings of unequal size, ∼2.5 and 3.5 nm in diameter, respectively, have been seen in atomic force microscopy images of the chloroplast ATPsynthase subunit III (proteolipid) oligomer (12Seelert H. Poetsch A. Dencher N.A. Engel A. Stahlberg H. Muller D.J. Nature. 2000; 405: 418-419Crossref PubMed Scopus (413) Google Scholar).Fig. 4 C shows contoured cross-sections of the three-dimensional model of the V0 at the positions indicated on the left in Fig. 4 A, image 1. Starting on the luminal side (bottom row, second image from the right) a peripheral density can be seen next to a ring-like structure (see arrow), which can be seen in the cross-sections going from the luminal to the cytoplasmic side. The density is ∼4 × 3 nm and could therefore accommodate a bundle of nine transmembrane α helices, consistent with this density being the membrane-bound C terminus of the a subunit. Although some densities within the proteolipid ring can be followed all the way through from the luminal to the cytoplasmic side (see arrowheads in Fig.4 C), the resolution of the model is not sufficient to define the number and arrangement of all the c subunit-like polypeptides in the proteolipid ring. An asymmetric protein ring comparable with the one shown here for the V-ATPase proton channel can be seen in electron microscopic images of the E. coli ATP synthase (29Böttcher B. Bertsche I. Reuter R. Gräber P. J. Mol. Bio. 2000; 296: 449-455Crossref PubMed Scopus (51) Google Scholar), again confirming that both enzymes share a similar overall architecture in their membrane domains.As mentioned above, the luminal side of the V0 channel shows two openings. The one that is covered on the luminal side by the spherical density seems to lead into the central channel formed by the proteolipids, while the other opening might lead into a cavity formed at the interface of the C-terminal domain of the a subunit and the outside of the c subunit ring. Such a solvent-accessible half-channel had been postulated for the interface of the F-ATPase a and c subunits (30Vik S.B. d'Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar). Higher up in the structure, the two channel openings combine into one large cavity without a clear indication for a boundary between proteolipids and the membrane-bound part of subunit a. It is conceivable that the resolution of the model is not sufficient to show all the details of the structure inside the channel, but it is also possible that the ring-like arrangement of the proteolipids is somewhat perturbed by the interaction with the membrane-bound domain of the a subunit. Proton translocation through the V0occurs via a protonation/deprotonation of lipid-exposed carboxyls in the C-terminal α helices of the c subunits (helix 3 of c“), most likely at the site of interaction with the a subunit. It has been shown by NMR spectroscopy that the E. coli F-ATPase proteolipid undergoes a large structural change when going from the protonated to the deprotonated state (31Rastogi V.K. Girvin M.E. Nature. 1999; 401: 200Google Scholar). The resulting structural asymmetry in the proteolipid ring might allow an intercalation of part of the a subunit transmembrane α helices into the ring as suggested by the cross-sections shown in Fig. 4 C.An additional cellular function for the V-ATPase membrane domain besides proton translocation has recently been reported based on a study with the yeast system. The data show that the free V0domain is involved in the late stage of membrane fusion and that this event involves a close contact between the cytoplasmic loops of the proteolipids of free V0 domains in the fusing vesicles (15Peters C. Bayer M. Bühler S. Andersen J. Mann M. Mayer A. Nature. 2001; 409: 581-588Crossref PubMed Scopus (421) Google Scholar). Calmodulin is then believed to interact with the proteolipids leading to a radial expansion of the proteolipid ring thus allowing the two bilayers to mix and fusion to occur. The structure here presented of the V0 shows that the cytoplasmic side of the proteolipid ring is covered by protein densities, most likely formed by the N-terminal domain of the a subunit and subunit d. This implies that in order for the c subunits of the opposing V0 domains to come in contact, a large structural change would have to occur. Such a conformational rearrangement might be induced by the interaction with calmodulin and/or other co-factors required for fusion. A large conformational change in the proteins covering the cytoplasmic domain of the V0 would also have to occur during binding of the V1 to enable the reported interaction of the N-terminal domain of a with the catalytic A subunits of the ATPase domain (32Landolt-Marticorena C. Williams K.M. Correa J. Chen W. Manolson M.F. J. Biol. Chem. 2000; 275: 15449-15457Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).While the structure presented here gives a first picture of the subunit arrangement in the V-ATPase membrane domain, studies at much higher resolution will be needed to be able to more fully understand the molecular mechanism of proton translocation in this class of enzymes. In eucaryotic cells, acidification and energization of organelles such as Golgi-derived vesicles, clathrin coated vesicles, synaptic vesicles, lysosomes, and the plant vacuole are accomplished by a vacuolar type ATPase (V-ATPase)1, and its proton pumping action plays a vital role in processes like protein trafficking, receptor-mediated endocytosis, neurotransmitter release, intracellular pH regulation, and waste management (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar, 2Finbow M.E. Harrison M.A. Biochem. J. 1997; 324: 697-712Crossref PubMed Scopus (230) Google Scholar, 3Nelson N. Harvey W.R. Physiol. Rev. 1999; 79: 361-385Crossref PubMed Scopus (367) Google Scholar). The vacuolar ATPase contains a large cytoplasmic ATPase domain (V1) and a membrane-embedded proton channel, V0. The two parts are connected via a stalk structure that functions to transmit the energy released during ATP hydrolysis taking place on the V1 to drive proton translocation across the membrane-bound V0. The eucaryotic V-ATPase is composed of 13 different polypeptides, with molecular masses ranging from 13 to 100 kDa. Eight of these, subunits A-H, form the cytoplasmic ATPase domain, while the membrane-embedded proton channel is made of the remaining five, subunits a, c, c′, c“ and d. A 14th subunit, Ac45, which is associated with the membrane-bound domain, is present in the mammalian enzyme in some tissues (4Supek F. Supekova L. Mandiyan S. Pan Y-C. Nelson H. Nelson N. J. Biol. Chem. 1994; 269: 24102-24106Abstract Full Text PDF PubMed Google Scholar). The V-ATPase is structurally and functionally related to the F1F0-ATPsynthase, and it is believed that both enzymes have evolved from a common ancestor (5Gogarten J.P. Kibak H. Dittrich P. Taiz L. Bowman E.J. Bowman B.J. Manolson M.F. Poole R.J. Date T. Oshima T. Konishi J. Denda K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6661-6665Crossref PubMed Scopus (531) Google Scholar). In case of the F-ATPase, a high resolution crystal structure exists for the cytoplasmic domain (6Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2733) Google Scholar), and based on the degree of sequence identity between the catalytic subunits of the F- and V-ATPase, it is likely that the V1-ATPase catalytic core has a similar three-dimensional-fold. The functional element proton channel is less well understood mainly because there is no detailed structure available for the intact membrane domain of either V- or F-ATPase. It is generally assumed that the c subunits are arranged in a ring-like fashion with the a subunit bound at the outside of the ring, and there is evidence for such an arrangement from electron microscopic images of both enzymes (7Wilkens" @default.
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W2130261095 date "2001-11-01" @default.
W2130261095 modified "2023-09-26" @default.
W2130261095 title "Three-dimensional Structure of the Vacuolar ATPase Proton Channel by Electron Microscopy" @default.
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