Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore

Sergio Couoh-Cardel; Yi Ching Hsueh; Stephan Wilkens; Liviu Movileanu

doi:10.1038/srep24774

Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore

Sergio Couoh-Cardel, Yi Ching Hsueh, Stephan Wilkens, Liviu Movileanu

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Abstract

The vacuolar H⁺ -ATPase (V-ATPase) is a rotary motor enzyme that acidifies intracellular organelles and the extracellular milieu in some tissues. Besides its canonical proton-pumping function, V-ATPase's membrane sector, V_o, has been implicated in non-canonical functions including membrane fusion and neurotransmitter release. Here, we report purification and biophysical characterization of yeast V-ATPase c subunit ring (c-ring) using electron microscopy and single-molecule electrophysiology. We find that yeast c-ring forms dimers mediated by the c subunits' cytoplasmic loops. Electrophysiology measurements of the c-ring reconstituted into a planar lipid bilayer revealed a large unitary conductance of ∼8.3 nS. Thus, the data support a role of V-ATPase c-ring in membrane fusion and neuronal communication.

Original language	English (US)
Article number	24774
Journal	Scientific reports
Volume	6
DOIs	https://doi.org/10.1038/srep24774
State	Published - Apr 21 2016

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title = "Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore",

abstract = "The vacuolar H+ -ATPase (V-ATPase) is a rotary motor enzyme that acidifies intracellular organelles and the extracellular milieu in some tissues. Besides its canonical proton-pumping function, V-ATPase's membrane sector, Vo, has been implicated in non-canonical functions including membrane fusion and neurotransmitter release. Here, we report purification and biophysical characterization of yeast V-ATPase c subunit ring (c-ring) using electron microscopy and single-molecule electrophysiology. We find that yeast c-ring forms dimers mediated by the c subunits' cytoplasmic loops. Electrophysiology measurements of the c-ring reconstituted into a planar lipid bilayer revealed a large unitary conductance of ∼8.3 nS. Thus, the data support a role of V-ATPase c-ring in membrane fusion and neuronal communication.",

author = "Sergio Couoh-Cardel and Hsueh, {Yi Ching} and Stephan Wilkens and Liviu Movileanu",

note = "Funding Information: Elena Milgrom is acknowledged for excellent technical assistance. We thank Avinash Kumar Thakur for his experimental assistance during the very early stage of this project and Dr. Rebecca Oot for the critical reading of the manuscript. This work was supported by NIH grants R01 GM058600 (to S.W.) and R01 GM088403 (to L.M.).",

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day = "21",

doi = "10.1038/srep24774",

language = "English (US)",

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journal = "Scientific reports",

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T1 - Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore

AU - Couoh-Cardel, Sergio

AU - Hsueh, Yi Ching

AU - Wilkens, Stephan

AU - Movileanu, Liviu

N1 - Funding Information: Elena Milgrom is acknowledged for excellent technical assistance. We thank Avinash Kumar Thakur for his experimental assistance during the very early stage of this project and Dr. Rebecca Oot for the critical reading of the manuscript. This work was supported by NIH grants R01 GM058600 (to S.W.) and R01 GM088403 (to L.M.).

PY - 2016/4/21

Y1 - 2016/4/21

N2 - The vacuolar H+ -ATPase (V-ATPase) is a rotary motor enzyme that acidifies intracellular organelles and the extracellular milieu in some tissues. Besides its canonical proton-pumping function, V-ATPase's membrane sector, Vo, has been implicated in non-canonical functions including membrane fusion and neurotransmitter release. Here, we report purification and biophysical characterization of yeast V-ATPase c subunit ring (c-ring) using electron microscopy and single-molecule electrophysiology. We find that yeast c-ring forms dimers mediated by the c subunits' cytoplasmic loops. Electrophysiology measurements of the c-ring reconstituted into a planar lipid bilayer revealed a large unitary conductance of ∼8.3 nS. Thus, the data support a role of V-ATPase c-ring in membrane fusion and neuronal communication.

AB - The vacuolar H+ -ATPase (V-ATPase) is a rotary motor enzyme that acidifies intracellular organelles and the extracellular milieu in some tissues. Besides its canonical proton-pumping function, V-ATPase's membrane sector, Vo, has been implicated in non-canonical functions including membrane fusion and neurotransmitter release. Here, we report purification and biophysical characterization of yeast V-ATPase c subunit ring (c-ring) using electron microscopy and single-molecule electrophysiology. We find that yeast c-ring forms dimers mediated by the c subunits' cytoplasmic loops. Electrophysiology measurements of the c-ring reconstituted into a planar lipid bilayer revealed a large unitary conductance of ∼8.3 nS. Thus, the data support a role of V-ATPase c-ring in membrane fusion and neuronal communication.

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M1 - 24774

ER -

Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore

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