Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

Couoh‐Cardel, Sergio; Milgröm, Elena; Wilkens, Stephan

doi:10.1074/jbc.m115.662494

Cited by 54 publications

(93 citation statements)

References 63 publications

(78 reference statements)

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“…4, a-c, showing the characteristic features as seen in earlier reconstructions of bovine (44) and yeast (38) V o , including the densities above the membrane (a NT and subunit d), the cleft between the density for a CT and the c-ring (Fig. 4a, arrowhead), which opens into a solvent (stain)-accessible pore as seen in the bottom view (Fig.…”

Section: Purification Of V O Membrane Sector and Reconstitution Intomentioning

confidence: 58%

“…Lipid Nanodiscs-We previously developed a procedure for purification of milligram amounts of yeast V-ATPase V o sector for functional and structural studies (38). Briefly, V o was solubilized from vacuolar membranes and affinity-captured via a calmodulin binding peptide fused to the C terminus of the vac- uole-specific isoform of subunit a (Vph1p).…”

Section: Purification Of V O Membrane Sector and Reconstitution Intomentioning

confidence: 99%

“…Besides glucose levels (30), V-ATPase assembly in animal cells can be induced by a variety of signals, including cell maturation (33) and stimulation by hormones (34) and growth factors (6). Upon enzyme dissociation, the activity of both sectors is silenced; that is, the V 1 no longer hydrolyzes MgATP (35,36), and the V o no longer translocates protons (37,38). Although studies in yeast suggest that V 1 activity silencing depends on the C-terminal domain of subunit H, possibly together with inhibitory MgADP (35,39,40), the mechanism by which passive proton transport across V o is blocked is less well understood.…”

mentioning

confidence: 99%

“…The structure of eukaryotic V-ATPase has been analyzed by EM, and together with crystal structures of individual subunits and subcomplexes from yeast V-ATPase and related bacterial enzymes, the EM reconstructions have allowed generation of pseudoatomic models of the intact enzyme (22,41,42) and its functional V 1 (43) and V o (38,44) sectors. Although the resulting structural models together with biochemical data provide valuable information on the mechanism of ATP hydrolysis-driven proton pumping, we only have a limited understanding of the mechanism of reversible enzyme dissociation and reassociation.…”

mentioning

confidence: 99%

“…Although the resulting structural models together with biochemical data provide valuable information on the mechanism of ATP hydrolysis-driven proton pumping, we only have a limited understanding of the mechanism of reversible enzyme dissociation and reassociation. We recently obtained a cryo-EM reconstruction of yeast V o (38), and although a comparison with EM models of holo V-ATPase showed that a NT undergoes a large structural change to bind the rotor subunit d in free V o , the resolution of the model was insufficient to precisely define the a NT -d interface.…”

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confidence: 99%

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Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Stam

Wilkens

2017

Journal of Biological Chemistry

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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...

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Section: Purification Of V O Membrane Sector and Reconstitution Intomentioning

confidence: 58%

Section: Purification Of V O Membrane Sector and Reconstitution Intomentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Stam

Wilkens

2017

Journal of Biological Chemistry

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Tender love and disassembly: How a TLDc domain protein breaks the V‐ATPase

et al. 2023

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Vacuolar ATPases (V-ATPases, V 1 V o -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V 1 -ATPase disengages from V o proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V 1 and V o subcomplexes.

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Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H⁺‐ATPase

Sharma

Wilkens

2017

Protein Science

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Vacuolar H1 -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. VATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1 -ATPase and membrane-integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V 1 V 0 ND). We show that V 1 V 0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.

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Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

Cited by 54 publications

References 63 publications

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Tender love and disassembly: How a TLDc domain protein breaks the V‐ATPase

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H⁺‐ATPase

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Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

Cited by 54 publications

References 63 publications

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Tender love and disassembly: How a TLDc domain protein breaks the V‐ATPase

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

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Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H⁺‐ATPase