V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

Yokoyama, Ken; Muneyuki, Eiro; Amano, Toyoki; Mizutani, Seiji; Yoshida, Masasuke; Ishida, Masami; Ohkuma, S.

doi:10.1074/jbc.273.32.20504

Cited by 70 publications

(83 citation statements)

References 45 publications

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“…Alternatively, the loss of activity is suggestive of product inhibition, which could also be an effective means of minimizing unproductive ATP hydrolysis in vivo. Similar behavior of the M. sexta cytosolic V 1 has been attributed to product inhibition (14), and a more detailed analysis of the V 1 -ATPase of Thermus thermophilus (44) has clearly demonstrated that this enzyme can be inactivated during ATP hydrolysis by entrapping an inhibitory MgADP at the catalytic site. In both of these cases, the similarity to entrapping of MgADP by F 1 -ATPases has been noted, but there are also some important differences between the behavior of the yeast cytosolic V 1 complexes and F 1 -ATPases (32,(35)(36)(37).…”

Section: Figmentioning

confidence: 84%

The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes

Parra

Keenan

Kane

2000

Journal of Biological Chemistry

156

204

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V-ATPases are composed of a peripheral complex containing the ATP-binding sites, the V 1 sector, attached to a membrane complex containing the proton pore, the V o sector. In vivo, free, inactive V 1 and V o sectors exist in dynamic equilibrium with fully assembled, active V 1 V o complexes, and this equilibrium can be perturbed by changes in carbon source. Free V 1 complexes were isolated from the cytosol of wild-type yeast cells and mutant strains lacking V o subunit c (Vma3p) or V 1 subunit H (Vma13p). V 1 complexes from wild-type or vma3⌬ mutant cells were very similar, and contained all previously identified yeast V 1 subunits except subunit C (Vma5p). These V 1 complexes hydrolyzed CaATP but not MgATP, and CaATP hydrolysis rapidly decelerated with time. V 1 complexes from vma13⌬ cells contained all V 1 subunits except C and H, and had markedly different catalytic properties. The initial rate of CaATP hydrolysis was maintained for much longer. The complexes also hydrolyzed MgATP, but showed a rapid deceleration in hydrolysis. These results indicate that the H subunit plays an important role in silencing unproductive ATP hydrolysis by cytosolic V 1 complexes, but suggest that other mechanisms, such as product inhibition, may also play a role in silencing in vivo. V-ATPases1 are highly conserved proton pumps distributed throughout the vacuolar network in all eukaryotic cells. VATPases maintain organelle acidification and affect cytosolic pH and ion balance, and their activity has been linked to a diverse array of cellular processes ranging from zymogen activation to protein sorting to viral membrane fusion events (1, 2). V-ATPases are comprised of two structural domains, the V 1 domain, which consists of a complex of peripheral subunits containing the nucleotide-binding sites attached to the cytoplasmic face of membrane, and the V o domain, which is comprised of several integral membrane and tightly associated peripheral proteins that contain the proton pore (1, 2). The yeast V-ATPase has at least eight V 1 subunits (designated A, B, C, D, E, F, G, and H) and five V o subunits (designated a, c, cЈ, cЉ, and d) (1,3,4 (10,13,14). Starvation appears to stimulate disassembly of V 1 from V o , but this disassembly is fully reversible upon refeeding (13-15). This reversible association between V 1 and V o is believed to regulate V-ATPase function in vivo: disassembly of V-ATPase complexes may conserve ATP when energy reserves are low and reassembly of the enzyme may provide the renewed proton pumping capacity necessary to prevent cytosolic acidification when active metabolism resumes (16,17).A constitutively active free V 1 in the cytosol could quickly become lethal to the cell by hydrolyzing cytosolic reserves of ATP. Graf et al. (14) have isolated cytosolic V 1 complexes from M. sexta and shown that these complexes exhibit Ca 2ϩ -dependent ATP hydrolysis at nonphysiological Ca 2ϩ concentrations but hydrolyze MgATP only in the presence of methanol. The properties of V 1 complexes have also been examined by recons...

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Section: Figmentioning

confidence: 84%

The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes

Parra

Keenan

Kane

2000

Journal of Biological Chemistry

156

204

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“…Aliquants of purified protein were frozen in liquid nitrogen and stored at −80°C. Because no nucleotide was added during the purification procedure, the preparation of enzyme is presumed to be in a nucleotidefree state (23,50). Purified V-ATPase showed >95% N,N'-dicyclohexylcarbodiimide (DCCD) sensitivity in ATPase assays (51).…”

Section: Methodsmentioning

confidence: 99%

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

Lau

Rubinstein

2010

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

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The eubacterium Thermus thermophilus uses a macromolecular assembly closely related to eukaryotic V-ATPase to produce its supply of ATP. This simplified V-ATPase offers several advantages over eukaryotic V-ATPases for structural analysis and investigation of the mechanism of the enzyme. Here we report the structure of the complex at ∼16 Å resolution as determined by single particle electron cryomicroscopy (cryo-EM). The resolution of the map and our use of cryo-EM, rather than negative stain EM, reveals detailed information about the internal organization of the assembly. We could separate the map into segments corresponding to subunits A and B, the threefold pseudosymmetric C-subunit, a central rotor consisting of subunits D and F, the L-ring, the stator subcomplex consisting of subunits I, E, and G, and a micelle of bound detergent. The architecture of the V O region shows a remarkably small area of contact between the I-subunit and the ring of L-subunits and is consistent with a two half-channel model for proton translocation. The arrangement of structural elements in V O gives insight into the mechanism of torque generation from proton translocation. membrane protein | single particle analysis V acuolar-type ATPases (V-ATPases) in eukaryotes function as ATP-driven proton pumps that acidify intracellular compartments including lysosomes, endosomes, and secretory vesicles. This acidification, in turn, affects diverse processes including protein sorting and degradation, overall ion homeostasis, and protection of cells from oxidative stress (1). Extracellular acidification by V-ATPases is linked to tumor invasion and metastasis and osteoporosis (2). F-type ATP synthases and V-type ATPases are evolutionarily related but differ in the details of subunit composition and arrangement. Both F-and V-type ATPases use a rotary catalytic mechanism where proton translocation through the membranebound F O or V O region, respectively, generates a torque on a rotor subcomplex that drives ATP synthesis in the F 1 or V 1 region. The enzymes can also run in the opposite direction with ATP hydrolysis in the F 1 or V 1 region resulting in proton pumping through F O or V O . This mechanism has been the subject of a large body of research for F-type ATP synthases (e.g., 3-5), but there has also been direct demonstration of rotary catalysis for V-ATPases (6). Some archaea and eubacteria use a complex more closely related to VATPase than F-type ATP synthase, sometimes called an A-ATPase, to generate their supply of ATP (7).The V-ATPase from the eubacterium Thermus thermophilus is composed of nine different subunits with a stoichiometry of A 3 B 3 CDE 2 FG 2 IL 12 (Fig. S1). Subunit nomenclature for this family of enzymes differs between F-and V-type complexes and from organism to organism. Where the eukaryotic ATP synthase F 1 catalytic region consists of α 3 β 3 γδε, the V-ATPase V 1 catalytic region consists of B 3 A 3 DF with no equivalent of the ε-subunit. The catalytic A-subunit of V-ATPase is homologous to the F-type ATP synthas...

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“…In eukaryotic cells, V 0 V 1 -ATPases exist in both intracellular compartments and plasma membranes, and are responsible for the acidification of intracellular compartments, renal acidification, born resorption, and tumor metastasis (2). On the other hand, most prokaryotic V 0 V 1 -ATPases produce ATP using the energy of a transmembrane proton electrochemical gradient that is generated by a respiratory chain (4,5).…”

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confidence: 99%

“…The V 0 V 1 -ATPase is capable of both ATP-driven proton translocation and proton-driven ATP synthesis and functions as ATP synthase in vivo (5,23 (Table I). Although the molecular size of some subunits of T. thermophilus is smaller than that of eukaryotic counterparts, each subunit of T. thermophilus shows a sequence similarity to its eukaryotic counterpart (see Table I).…”

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confidence: 99%

Subunit Arrangement in V-ATPase from Thermus thermophilus

Yokoyama¹,

Nagata

Imamura³

et al. 2003

Journal of Biological Chemistry

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The V 0 V 1 -ATPase of Thermus thermophilus catalyzes ATP synthesis coupled with proton translocation. It consists of an ATPase-active V 1 part (ABDF) and a proton channel V 0 part (CLEGI), but the arrangement of each subunit is still largely unknown. Here we found that acid treatment of V 0 V 1 -ATPase induced its dissociation into two subcomplexes, one with subunit composition ABDFCL and the other with EGI. Exposure of the isolated V 0 to acid or 8 M urea also produced two subcomplexes, EGI and CL. Thus, the C subunit (homologue of d subunit, yeast Vma6p) associates with the L subunit ring tightly, and I (homologue of 100-kDa subunit, yeast Vph1p), E, and G subunits constitute a stable complex. Based on these observations and our recent demonstration that D, F, and L subunits rotate relative to A 3 B 3 (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. V 0 V 1 -ATPases (V-ATPases) are the ATPase/ATP synthase superfamily that catalyzes the exchange of the energy between proton translocation across membranes and the energy of ATP hydrolysis/synthesis (1-3). They are widely distributed in different types of eukaryotic cells and some bacteria (2, 4). In eukaryotic cells, V 0 V 1 -ATPases exist in both intracellular compartments and plasma membranes, and are responsible for the acidification of intracellular compartments, renal acidification, born resorption, and tumor metastasis (2). On the other hand, most prokaryotic V 0 V 1 -ATPases produce ATP using the energy of a transmembrane proton electrochemical gradient that is generated by a respiratory chain (4, 5).The overall structure of V 0 V 1 -ATPases is similar to that of F 0 F 1 -ATPases (␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 a 1 b 2 c 10Ϫ14 ), which are responsible for ATP synthesis in mitochondria, chloroplast, and plasma membranes of eubacteria (3, 6). Both are composed of two functional domains, the peripheral catalytic V 1 or F 1 and a membrane embedded ion translocating domain called V 0 or F 0 .The structure and subunit arrangements of F 0 F 1 -ATPases are well characterized. The x-ray structure of F 1 revealed a hexamer of alternating ␣ and ␤ surrounding a central cavity containing a highly ␣-helical ␥ subunit (7). The ␥ and ⑀ subunit constituted a central shaft, which directly contacted with the c subunit ring in F 0 (8). The b subunit has a hydrophobic Nterminal domain anchored in the membrane, and a hydrophilic C-terminal domain forms an elongated peripheral stalk that interacts with the F 1 moiety as a stator (9). The a subunit in F 0 , which consists of a stator part together with b subunits, is situated peripherally to the c subunit ring and plays a crucial role in the proton translocation (3, 10, 11).Like the F 0 F 1 -ATPases, the peripheral V 1 part contains a catalytic core, which is composed of three copies each of A and B subunits. The A subunit contains a catalytic site, and the A and B subunits are arranged alternately, forming a hexameric cylinder. The D subunit, which fills the central cavity of the A 3 B 3 cylinder...

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V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

Cited by 70 publications

References 45 publications

The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes

The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

Subunit Arrangement in V-ATPase from Thermus thermophilus

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V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

Cited by 70 publications

References 45 publications

The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes

The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V O motor

Subunit Arrangement in V-ATPase from Thermus thermophilus

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Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor