Crystal structure of yeast V<sub>1</sub>‐<scp>ATP</scp>ase in the autoinhibited state

Oot, Rebecca A.; Kane, Patricia M.; Berry, Edward A.; Wilkens, Stephan

doi:10.15252/embj.201593447

Cited by 49 publications

(103 citation statements)

References 55 publications

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“…A comparison with the recent EM reconstructions of yeast V-ATPase in three states (22) suggests that, upon enzyme dissociation, free V o is halted in state 3. We showed previously that autoinhibited, membrane-detached V 1 -ATPase is halted in state 2 (45), and we propose that this conformational mismatch to state 3 V o could function to prevent unintended reassembly of holo V-ATPase under conditions when the proton pumping activity of the enzyme is not needed.…”

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confidence: 71%

“…Recently, we determined the 6.2-to 6.5-Å crystal structure of autoinhibited yeast V 1 -ATPase (40). Interestingly, a comparison of the structure of autoinhibited V 1 with the structure of V 1 as part of holo V-ATPase (22) revealed that the membranedetached V 1 is halted in state 2 based on the rotational position of the DF rotor relative to the inhibitory H subunit (40).…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, a comparison of the structure of autoinhibited V 1 with the structure of V 1 as part of holo V-ATPase (22) revealed that the membranedetached V 1 is halted in state 2 based on the rotational position of the DF rotor relative to the inhibitory H subunit (40). The observation that autoinhibited V 1 is halted in state 2 together with the findings presented here that free V o is halted in state 3 indicates that there is a conformational mismatch between the two complexes as a result of regulated enzyme disassembly.…”

Section: Discussionmentioning

confidence: 99%

“…Upon enzyme dissociation, the activity of both sectors is silenced; that is, the V 1 no longer hydrolyzes MgATP (35,36), and the V o no longer translocates protons (37,38). Although studies in yeast suggest that V 1 activity silencing depends on the C-terminal domain of subunit H, possibly together with inhibitory MgADP (35,39,40), the mechanism by which passive proton transport across V o is blocked is less well understood.…”

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Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Stam

Wilkens

2017

Journal of Biological Chemistry

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Edited by Norma Allewell The vacuolar His a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells, where it acidifies the lumen of subcellular organelles, including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles (1-4). V-ATPase function is essential for pH and ion homeostasis (2), protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR) (5, 6), and Notch (7) signaling as well as hormone secretion (8) and neurotransmitter release (9). In animals, V-ATPase can also be found in the plasma membrane of polarized cells, where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation (1). The essential nature of eukaryotic V-ATPase is highlighted by the fact that complete loss of V-ATPase activity in animals is embryonic lethal (10). On the other hand, partial loss of enzyme function (or hyperactivity) has been associated with numerous widespread human diseases, including, but not limited to, renal tubular acidosis (11), osteoporosis (12), neurodegeneration (13), male infertility (14), deafness (15), diabetes (8), and cancer (16). Furthermore, V-ATPase is targeted by pathogens such as Mycobacterium tuberculosis or Legionella pneumophila (17, 18) to facilitate pathogen entry and survival. Because of its essential nature and key role in so many human diseases, VATPase has been identified as a potential drug target (19 -21).V-ATPase can be divided into a soluble catalytic sector, V 1 , and a membrane-integral proton channel sector, V o (Fig. 1). In the yeast Saccharomyces cerevisiae, V 1 is composed of eight different polypeptides, AB(C)DEFGH, that are arranged in an A 3 B 3 catalytic hexamer with a central stalk made of DF and three peripheral stators (EG heterodimers), one of which binds the single-copy H subunit. The ϳ320-kDa V o contains subunits acc'c''de, which are organized in a membrane-integral "proteolipid" ring (c 8 c'c' ' (22, 23)), a membrane-bound subunit a with an integral C-terminal domain (a CT ) that is bound at the periphery of the proteolipid ring, and an N-terminal cytoplasmic domain (a NT ) that is bound to subunit d (Fig. 1). The stoichiometry, location, and function of subunit e are not known. Eukaryotic V-ATPase belongs to the family of energy-transducing rotary motor ion pumps that also includes F 1 F o -ATP synthase, archaeal A-ATPase, and bacterial A/V-like ATPase (24,25). In V-ATPase, ATP hydrolysis at three catalytic sites in the A 3 B 3 hexamer is coupled to proton translocation via rotation of V 1 subunits DF that are connected to the subunit d-proteolipid ring subcomplex of V o . Proton translocation is through two aqueous half-channels at the interface of a CT and the prote-* This work was supported by National Institutes of Health Grant GM058600 (to S. W.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Na...

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confidence: 71%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Stam

Wilkens

2017

Journal of Biological Chemistry

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“…Thus, nucleotide-free V 1 -ATPases seem to form the catalytic dwell state, rather than the ATP-binding dwell and ADP-release dwell states. Recently, the crystal structure of S. cerevisiae V 1 -ATPase has been obtained at a 6.2 Å resolution49. This structure appears to be an inhibitory state wherein the subunit H inhibits the ATPase activity by stabilizing ADP binding to the catalytic site.…”

Section: Discussionmentioning

confidence: 99%

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

et al. 2016

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V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model.

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Tender love and disassembly: How a TLDc domain protein breaks the V‐ATPase

et al. 2023

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Vacuolar ATPases (V-ATPases, V 1 V o -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V 1 -ATPase disengages from V o proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V 1 and V o subcomplexes.

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Crystal structure of yeast V₁‐ATPase in the autoinhibited state

Cited by 49 publications

References 55 publications

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

Tender love and disassembly: How a TLDc domain protein breaks the V‐ATPase

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Crystal structure of yeast V1‐ATPase in the autoinhibited state

Cited by 49 publications

References 55 publications

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

Tender love and disassembly: How a TLDc domain protein breaks the V‐ATPase

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Crystal structure of yeast V₁‐ATPase in the autoinhibited state