Interacting Helical Surfaces of the Transmembrane Segments of Subunits a and c′ of the Yeast V-ATPase Defined by Disulfide-mediated Cross-linking

Kawasaki-Nishi, Shoko; Nishi, Tsuyoshi; Forgac, Michael

doi:10.1074/jbc.m308026200

Cited by 49 publications

(41 citation statements)

References 81 publications

(77 reference statements)

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“…Most of the amino acids identified are in the ring-contacting α-helices 7 and 8, except for two highly conserved membrane-embedded aspartate residues: D425 on the short linker connecting α-helices 1 and 2, and D481 on α-helix 3. In addition, cysteine-mediated cross-linking has helped define the location of specific a subunit residues in relation to the c′ subunit (42). These cross-linking results also agree well with the proposed a subunit model (Fig.…”

Section: Resultssupporting

confidence: 77%

“…Other than these differences, comparison of the two states reveals only minor conformational changes in individual subunits, suggesting little flexibility in the enzyme as a whole (Movie S3). The T. thermophilus V/A-ATPase thus seems to be relatively rigid compared with the S. cerevisiae V-ATPase (8) Numerous studies, many by Forgac and coworkers, have investigated the topology, function, and subunit arrangement of the S. cerevisiae V-ATPase V O region (31,34,(38)(39)(40)(41)(42)(43)(44). The model of the a subunit presented here is in excellent agreement with these studies.…”

Section: Resultssupporting

confidence: 72%

“…(C) Cysteine-mediated cross-linking (red lines) of one c′ subunit (magenta) to subunit a (green) (42). (D) Residues that, when mutated (blue spheres) in the c subunit (T32, T39, L50, I54, V57, G61, L131, F135, L139, and Y142) and a subunit (E721, L724, and N725), give partial resistance to bafilomycin (43,44).…”

Section: Discussionmentioning

confidence: 99%

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Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

Schep

Zhao

Rubinstein

2016

Proc. Natl. Acad. Sci. U.S.A.

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Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membraneembedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.cryo-EM | evolutionary covariance | V-ATPase | V/A-ATPase | structure V acuolar H + -ATPases (V-ATPases) are large membrane protein complexes responsible for the acidification of intracellular compartments in eukaryotes. These complexes are essential for processes including receptor-mediated endocytosis, coupled transport, and lysosomal degradation (1). V-ATPases localize to the plasma membrane of some specialized cells where they participate in bone resorption by osteoclasts (2) and urine acidification by the α-intercalated cells of the kidney (3). Malfunction or misregulation of these enzymes results in numerous disorders, including osteopetrosis (4) and renal tubular acidosis (5, 6). Expression of V-ATPases on the surfaces of some tumor cells results in increased tumor invasion and metastasis (7). The V/A-ATPase from the thermophilic eubacterium Thermus thermophilus is homologous to the eukaryotic V-ATPase and has a similar overall structure, but is smaller and simpler and functions in vivo as an ATP synthase. V-and V/A-ATPases have similar subunit folds and arrangements of subunits (8-11). The simplified architecture and superior stability of the T. thermophilus V/A-ATPase make it an ideal model to study the structure and function of V-ATPases. Both enzymes are composed of a soluble V 1 catalytic region and a membrane-embedded V O region. The V/A-ATPase subunits I, L, and C in T. thermophilus are homologous to the a, c, and d subunits in eukaryotic V-ATPases, respectively, and, for clarity, the eukaryotic subunit names are used here. With this convention, the T. thermophilus V/A-ATPase contains subunits A 3 B 3 DE 2 FG 2 ac 12 d whereas the eukaryot...

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Section: Resultssupporting

confidence: 77%

Section: Resultssupporting

confidence: 72%

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Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

Schep

Zhao

Rubinstein

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Topological studies suggest that the C-terminal domain of subunit a contains nine transmembrane helices (25), with the critical Arg 735 located in TM7 (23). Cross-linking studies have demonstrated that TM7 of subunit a is in close proximity to TM4 of subunit cЈ and TM2 of subunit cЉ (30,31), both of which contain an essential glutamic acid residue (21). Moreover, the data are consistent with swiveling of helices within both subunits a and the proteolipids relative to each other.…”

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confidence: 72%

Subunit a of the Yeast V-ATPase Participates in Binding of Bafilomycin

Wang

Inoué

Forgac

2005

Journal of Biological Chemistry

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The vacuolar (H ϩ )-ATPases (or V-ATPases) 2 are ATP-dependent proton pumps widely distributed among intracellular and plasma membranes of eukaryotic cells (1-11). They carry out ATP-driven transport of protons from the cytoplasm to either the lumen of internal compartments or to the extracellular space. V-ATPases present in intracellular membranes are important for membrane traffic processes such as receptor-mediated endocytosis and intracellular targeting of newly synthesized lysosomal enzymes (1). They also provide the acidic environment required for processing and degradation of macromolecules and the entry of certain envelope viruses and bacterial toxins, including anthrax toxin (12), as well as the driving force for coupled transport of small molecules, such as neurotransmitters. Plasma membrane V-ATPases function in a wide variety of normal and disease processes, including renal acidification, bone resorption, K ϩ secretion, sperm maturation, and tumor cell invasion (1, 7-11). V-ATPase inhibitors are thus potentially useful therapeutic agents for the treatment of a number of human diseases, including viral infection, osteoporosis, and cancer. V-ATPases are multisubunit complexes composed of two domains (1-9). The peripheral V 1 domain is composed of eight subunits (A-H) and functions to hydrolyze ATP. The integral V 0 domain is composed of six subunits (in yeast these are subunits a, c, cЈ, cЉ, d, and e) and is responsible for proton transport. The V-ATPases thus structurally resemble the ATP synthases (or F-ATPases) of mitochondria, chloroplasts, and bacteria that function in the synthesis of ATP (13-16).Like the F-ATPases (13-16), the V-ATPases have been shown to operate by a rotary mechanism (17, 18). ATP hydrolysis in the V 1 domain drives rotation of a central stalk composed of subunits D and F (17, 19). These subunits are linked to a ring of proteolipid subunits (c, cЈ, and cЉ) via subunit d (20). Each proteolipid subunit contains a single buried acidic residue that undergoes reversible protonation and deprotonation during the rotational catalysis (21). As the ring of proteolipid subunits rotates relative to subunit a, each buried carboxyl group on the ring is thought to pick up a proton from the cytoplasmic compartment via a hemi-channel located in subunit a (1, 15, 22, 23). Rotation of the ring brings this protonated carboxyl into contact with a second a subunit hemi-channel leading to the luminal or extracellular side of the membrane. Interaction between the carboxyl group and the positively charged guanidinium group of Arg 735 of subunit a (23) then causes release of the proton into the luminal hemi-channel.Subunit a is a 100-kDa integral membrane protein containing two domains (24). The N-terminal hydrophilic domain has a molecular mass of 50 kDa and is oriented toward the cytoplasmic side of the membrane (25). Together with subunits C, E, G, H, and the non-homologous domain of subunit A (19, 26 -29), the N-terminal domain forms a peripheral stalk or stator that keeps the catalytic domain of t...

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“…By contrast, His-743 is predicted to be near the lumenal border of TM7 and may therefore contribute to a lumenal hemichannel. The location of Arg-735 within the membrane is supported by the ability to form zero-length cross-links between cysteine residues introduced near Arg-735 in subunit a and cysteine residues near the critical buried glutamic acid residues in TM4 of subunit cЈ or TM3 of subunit cЉ (40,41).…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Membrane Topology of Transmembrane Segments in the C-terminal Hydrophobic Domain of the Yeast Vacuolar ATPase Subunit a (Vph1p) by Chemical Modification

Wang

Toei

Forgac

2008

Journal of Biological Chemistry

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The integral V 0 domain of the vacuolar (H ؉ )-ATPases (VATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V 0 that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEGmal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.2 are ATP-dependent proton pumps present in a variety of intracellular compartments, including endosomes, lysosomes, Golgi-derived vesicles, and secretory vesicles (1-3). Acidification of intracellular compartments is important for membrane traffic processes, protein degradation and processing, coupled transport of small molecules, and the entry of various pathogens, including envelope viruses like influenza virus and bacterial toxins like anthrax toxin (4). V-ATPases are also present at the plasma membrane of a variety of cell types, including renal-intercalated cells, osteoclasts, macrophages, and neutrophils, epididymal clear cells, insect goblet cells, and certain tumor cells (5-10). Plasma membrane V-ATPases play a critical role in processes such as urinary acidification, bone resorption, sperm maturation, pH homeostasis, coupled transport, and tumor metastasis.The V-ATPases are multisubunit complexes containing two domains (1-3). The V 1 domain is peripheral to the membrane, contains eight different polypeptides (subunits A-H) and carries out ATP hydrolysis. The V 0 domain is membrane-integral, contains subunits a, c, cЈ, cЉ, d, and e (in yeast), and is responsible for proton transport. Both the proteolipid subunits (c, cЈ, and cЉ) and subunit a contain residues that are essential for proton translocation (11, 12). The proteolipid subunits form a ring containing buried glutamic acid residues (13, 14) that are thought to undergo reversible protonation du...

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Interacting Helical Surfaces of the Transmembrane Segments of Subunits a and c′ of the Yeast V-ATPase Defined by Disulfide-mediated Cross-linking

Cited by 49 publications

References 81 publications

Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

Subunit a of the Yeast V-ATPase Participates in Binding of Bafilomycin

Analysis of the Membrane Topology of Transmembrane Segments in the C-terminal Hydrophobic Domain of the Yeast Vacuolar ATPase Subunit a (Vph1p) by Chemical Modification

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