The C-H Peripheral Stalk Base: A Novel Component in V1-ATPase Assembly

Zacariah L Hildenbrand,Sudheer K Molugu,Ricardo A Bernal,Daniela Stock,Maxim Antopolsky

doi:10.1371/journal.pone.0012588

Zacariah L Hildenbrand, Sudheer K Molugu + Show 3 more

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https://doi.org/10.1371/journal.pone.0012588

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Abstract

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V1Vo-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V1-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE3G3H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembly mechanism of the free V1-ATPase.

Highlights

Vacuolar ATPases (V-ATPases) are biological rotary motors that harness the energy derived from ATP hydrolysis to drive the translocation of protons across a membrane
In all V-ATPases the catalytic activity arises from conformational changes resulting from ATP-binding and hydrolysis on the A subunits near the AB interface [32,33,34].These changes drive the rotation of the heterodimeric DF central stalk which results in the translocation of protons across the membrane through the a(cc9)4-5c0d proteolipid ring [35,36,37]
The only situation in which the DF central stalk and the A3B3 catalytic complex could be captured in a single predominant orientation is when the two entities are held motionless by some coherent interaction as seen in our A3B3DF reconstruction

Summary

Introduction

Vacuolar ATPases (V-ATPases) are biological rotary motors that harness the energy derived from ATP hydrolysis to drive the translocation of protons across a membrane. These proton pumps generate electrochemical gradients across organelle and plasma membranes to facilitate a number of secondary transport systems that are involved in a wide variety of biological processes [1]. The soluble V1-domain has a molecular mass of approximately 640 kDa and is composed of eight subunits denoted A-H that are architecturally arranged into sub-complexes according to their distinct roles in the rotary mechanism. Subunits A and B form the A3B3 catalytic complex that is responsible for hydrolyzing ATP and inducing the rotation of the DF central rotor stalk

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