Functional reconstitution of vacuolar H+-ATPase from Vo proton channel and mutant V1-ATPase provides insight into the mechanism of reversible disassembly

Sharma, Stuti; Oot, Rebecca A.; Khan, Md. Murad; Wilkens, Stephan

doi:10.1074/jbc.ra119.007577

Cited by 22 publications

(42 citation statements)

References 51 publications

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“…1 ). The V 1 and V o domains of V-ATPase can assemble and disassemble in vivo depending on various stimuli, such as growth factors, amino acid starvation, and increased glucose concentration 13 , 14 ; therefore, the V-ATPase activity can be shut down and turned on like a switch.…”

Section: Introductionmentioning

confidence: 99%

Molecular basis of V-ATPase inhibition by bafilomycin A1

et al. 2021

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Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7’-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.

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Section: Introductionmentioning

confidence: 99%

Molecular basis of V-ATPase inhibition by bafilomycin A1

et al. 2021

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“…The eukaryotic V-ATPase is regulated by a unique mechanism involving dissociation/association of V 1 , that is likely to be a key factor in controlling the pH of acidic vesicles ( Kane and Parra, 2000 ; Sharma et al, 2019 ; Toei et al, 2010 ). In yeast, glucose depletion in the culture medium induces dissociation of the V 1 domain from V o domain, resulting in reduced proton pumping activity of the V-ATPase ( Figure 1—figure supplement 1A ).…”

Section: Introductionmentioning

confidence: 99%

Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance

Kishikawa

Nakanishi

Furuta

et al. 2020

eLife

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V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 domain, with proton flow through the Vo membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo domain adopts the auto-inhibited form and how the holo complex prevents formation of the inhibited Vo form.

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“…Three independent RAVE-V 1 preparations gave an average K d of 18.1 ± 4.8 nM by BLI. Interestingly, this affinity is lower than the observed affinity of isolated V 1 for subunit C (0.7 nM) but higher than the affinity of subunit C for isolated EG stalk complexes (42 nM) (32,(36)(37)(38). To address the contribution of the RAVE complex to the interaction between subunit C and RAVE-V 1 , we repeated the binding experiments with RAVE alone.…”

Section: Figure 2 Purification Of Rave-v 1 and Rave Complexesmentioning

confidence: 93%

“…To address this issue, we assessed whether RAVE-V 1 was able to bind to subunit C in vitro. We utilized biolayer interferometry (BLI) to quantitate binding between bacterially expressed maltose-binding protein (MBP)-tagged subunit C (MBP-C) and purified RAVE-V 1 in a strategy similar to that of the study by Sharma et al (29,32). Briefly, MBP-C was bound to BLI sensors loaded with anti-MBP antibody and then dipped into wells containing varied concentrations of RAVE-V 1 .…”

Section: Figure 2 Purification Of Rave-v 1 and Rave Complexesmentioning

confidence: 99%

“…RAVE–V 1 in combination with exogenous C subunit was unable to assemble with intact V o complexes reconstituted into lipid nanodiscs. However, purified RAVE accelerates assembly of active and concanamycin-sensitive V-ATPases when combined with a V 1 complex containing a mutant H subunit incapable of assuming inhibitory conformation ( 6 , 32 ) along with subunit C and V o -containing nanodiscs. Taken together, these results suggest that RAVE–V 1 is a stable intermediate complex that acquires subunit C in the process of V-ATPase reassembly in vivo .…”

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confidence: 99%

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Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes

Jaskolka

Tarsio

Smardon

et al. 2021

Journal of Biological Chemistry

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Edited by Henrik DohlmanThe vacuolar H + -ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V 1 domain from the integral membrane V o domain, accompanied by release of the V 1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H + -ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V 1 complexes. Both purified RAVE and RAVE-V 1 contain substoichiometric levels of subunit C. RAVE-V 1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V 1 interacts with the N-terminal domain of V o subunit Vph1 in vitro. RAVE-V 1 complexes, like isolated V 1 , have no MgATPase activity, suggesting that RAVE cannot reverse V 1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V 1 carrying a mutant subunit H incapable of inhibition with V o complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.

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Functional reconstitution of vacuolar H+-ATPase from Vo proton channel and mutant V1-ATPase provides insight into the mechanism of reversible disassembly

Cited by 22 publications

References 51 publications

Molecular basis of V-ATPase inhibition by bafilomycin A1

Molecular basis of V-ATPase inhibition by bafilomycin A1

Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance

Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes

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