Breaking up and making up: The secret life of the vacuolar H⁺‐ATPase

Abstract

The vacuolar ATPase (V-ATPase; V 1 V o -ATPase) is a large multisubunit proton pump 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. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.

Role of the H subunit C-terminal domain in the assembly of the vacuolar H+-ATPase

“…S1E-G) (16,(29)(30)(31). Moreover, a comparison of the cryoEM models of the three rotary states of holo V-ATPase (28) with the structures of autoinhibited V1 (32) and Vo (29,31) showed that while V1 is halted in state 2, free Vo adopted state 3 (10) (Supplementary Fig. S1).…”

“…S1). From this observation, we hypothesized that the state mismatch together with the large conformational changes of HCT and aNT that accompany enzyme dissociation explain why V1 does not readily bind free Vo under Role of subunit H in V-ATPase assembly physiological conditions in vitro (10,33), a safety-mechanism that likely evolved to prevent spontaneous reassembly in vivo when the disassembled state is required.…”

“…V-ATPase is a large (~ 1 MDa) membrane protein complex that can be divided into a cytosolic V1-ATPase and a membrane integral Vo proton channel. In yeast, V1 and Vo are made of A3B3(C) DE3FG3H and ac8c'c"def, respectively (10). ATP hydrolysis takes place at three out of six A-B interfaces of the catalytic A3B3 hexamer.…”

Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2: Implication for regulation of V-ATPase, CTH2 function and rational drug design

“…In order to accomplish an efficient transfer of 4 ATP hydrolysis energy to c-ring rotation, the rotation of A 3 B 3 headpiece is prevented by three peripheral stalks (Zhang et al, 2008). Each of these stalks is composed by a G/E heterodimer, which anchors the A 3 B 3 headpiece to the membrane through either: i) direct binding to the N-terminal tail of a-subunit, or ii) indirectly through subunit C. In particular, while two of three G/E-stalks are directly bound to a-subunit, the third G/Estalk is bound to subunit C, which itself interacts simultaneously with a-subunit and the second G/E-peripheral stalk (Oot & Wilkens, 2012;Oot et al, 2017;Zhang et al, 2008).…”

“…It is noteworthy that during the disassembly of V 1 -sector from V O -sector, the proteinprotein interactions between a-subunit and G/E-stalks as well as C-subunit and G/E-stalk are destabilized through a yet unknown molecular mechanism (Oot et al, 2017).…”