V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 domain rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here, we report the atomic models of 18 catalytic intermediates of the V1 domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

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.

Microfluidic devices have emerged as a new cell culture tool, which can mimic the structure and physiology of living human organs. However, no standardized culture method for a microfluidic device has yet been established. Here, we describe the effects of various conditions on cell proliferation in a microchannel with a depth smaller than 100 μm. Primary endothelial cell proliferation was suppressed with a decrease in the culture medium volume per cell culture area. Moreover, cell growth was compared with or without medium flow, and the optimum culture condition was determined to be 1 μL/h flow in a 65-μm-deep microchannel. In addition, glucose consumption was greater under fluidic conditions than under static conditions, and the ability of tumor (HeLa) cells to convert glucose into lactate appeared to be higher in a static culture than that in a fluidic culture. Overall, our results will serve as a useful guide for designing a microfluidic cell culture platform in a channel smaller than 100 μm.

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 moiety rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here we have determined the atomic models of 18 catalytic intermediates of the V1 moiety of V/A-ATPase under different reaction conditions by single particle Cryo-EM, which revealed that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis 2 in the hydrophilic V1 moiety, with proton flow through the Vo membrane moiety, via 3 rotation of the central rotor complex relative to the surrounding stator apparatus. Upon 4 dissociation from the V1 domain, the Vo of eukaryotic V-ATPase can adopt a 5 physiologically relevant auto-inhibited form in which proton conductance through the Vo 6 is prevented, however the molecular mechanism of this inhibition is not fully understood. 7Using cryo-electron microscopy, we determined the structure of both the holo V/A-8ATPase and the isolated Vo at near-atomic resolution, respectively. These structures 9 clarify how the isolated Vo adopts the auto-inhibited form and how the holo complex 10 prevents the formation of this inhibited Vo form. 11 12 Short Title: The switching mechanism of rotary V-ATPase 13 One Sentence Summary: Cryo-EM structures of rotary V-ATPase reveal the ON-OFF 14 switching mechanism of H + translocation in the Vo membrane domain. 15 16 17 Main Text: 1 Rotary ATPase/ATP synthases, roughly classified into F type and V type 2 ATPase, are marvelous, tiny rotary machines (1-5). These rotary motor proteins share a 3 basic molecular architecture composed of a central rotor complex and the surrounding 4 stator apparatus. These proteins function to couple ATP hydrolysis/synthesis in the 5 hydrophilic F1/V1 moiety with proton translocation through the membrane embedded 6 hydrophobic Fo/Vo moiety by rotation of the central rotor complex relative to surrounding 7 stator apparatus, via a rotary catalytic mechanism (Figure 1) (2-6). 8 Thus, both F and V type ATPases are capable of either ATP synthase coupled 9with proton motive force driven by membrane potential or proton pumping powered by 10