LAUSR

In this work, we show the first purification and functional characterization of the proteolipid ring (c-ring) complex of the membrane-bound Vo sector of the eukaryotic vacuolar H+-ATPase (V-ATPase) from the yeast Saccharomyces cerevisiae using techniques of membrane biochemistry, structural biology, and single-molecule electrophysiology. Our work was motivated by, and builds on, a large body of literature spanning more than three decades that suggested that Vo, besides its proton pumping function in holo V-ATPase, has other, so-called “non-canonical” functions in essential processes, such as membrane fusion and neurotransmitter release. However, direct evidence that Vo, and specifically, its core proteolipid ring (c-ring) complex, possesses the required properties to fulfill these non-canonical functions is still lacking. Our results presented here show that the c-ring forms a large-conductance transmembrane pore, which inserted into planar lipid bilayers with a preferred orientation. Moreover, using single-particle cryo-electron microscopy, we demonstrate that the purified c-ring forms dimers through an interaction mediated by the cytoplasmic loops of the c subunits. Taken together, these findings provide the first direct attestation in support of the proposed non-canonical functions of the V-ATPase Vo sector.1 This work was supported by NIH grants R01 GM058600 (to S.W.) and R01 GM088403 (to L.M.). 1. Couoh-Cardel, S.; Hsueh, Y. C.; Wilkens, S.; Movileanu, L., Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore. Scientific reports 2016, 6, 24774.