Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Abstract

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...

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

“…1, D and E). Examination of purified V o ND using negative stain EM showed single particles of V o ND with the typical size and appearance as described previously (26) (Fig. 1F).…”

“…At the same time, a NT moves from its peripheral position near C foot and EG in V 1 V o (Fig. 1A) toward a more central position in free V o to bind subunit d (18,(25)(26)(27) (Fig. S1, E-G).…”

Regulation of V-ATPase Activity and Organelle pH by Phosphatidylinositol Phosphate Lipids

“…Potential PIP binding mutations were designed as described above, and a single point mutation (Figure 4) in the proximal domain of Stv1NT almost completely abolished flotation of Stv1NT with PI(4)Pcontaining liposomes in vitro (Banerjee and Kane, 2017). This (Zhao et al, 2015), and a disassembled and autoinhibited conformation in the isolated V o sector, colored in green (Stam and Wilkens, 2017). The chain traces of the c-ring comprized of c8c c is colored in gray and the spherical atoms of the d-subunit is colored in blue.…”

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly

“…ferent rotational states: V 1 in state 2 and V o in state 3 (22,52,53). This suggests that the MgATPase activity that is necessary for efficient disassembly serves to generate the rotational state mismatch associated with enzyme dissociation, a mismatch that likely functions to prevent rebinding of V 1 to V o under cellular conditions that favor the disassembled state.…”