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福利彩票开奖快乐十分:Structural comparison of the vacuolar and Golgi V-ATPases from Saccharomyces cerevisiae
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Proton-pumping V-ATPases maintain the pH of intracellular compartments in all eukaryotic cells and the extracellular environment of specialized cells. V-ATPase dysfunction is associated with diseases including osteopetrosis, renal tubular acidosis, and cancer. Many subunits of mammalian V-ATPases exist as multiple isoforms that are expressed in an organelle-, cell-, or tissue-dependent manner, but the consequences of this diversity are not understood. In the yeast Saccharomyces cerevisiae, there are only two V-ATPase isoforms, making it an ideal system to study isoform-specific characteristics. Identifying similarities and differences in structure and function between V-ATPase isoforms improves our understanding of the enzyme’s diverse cellular functions.
Proton-translocating vacuolar-type ATPases (V-ATPases) are necessary for numerous processes in eukaryotic cells, including receptor-mediated endocytosis, protein maturation, and lysosomal acidification. In mammals, V-ATPase subunit isoforms are differentially targeted to various intracellular compartments or tissues, but how these subunit isoforms influence enzyme activity is not clear. In the yeast Saccharomyces cerevisiae, isoform diversity is limited to two different versions of the proton-translocating subunit a: Vph1p, which is targeted to the vacuole, and Stv1p, which is targeted to the Golgi apparatus and endosomes. We show that purified V-ATPase complexes containing Vph1p have higher ATPase activity than complexes containing Stv1p and that the relative difference in activity depends on the presence of lipids. We also show that VO complexes containing Stv1p could be readily purified without attached V1 regions. We used this effect to determine structures of the membrane-embedded VO region with Stv1p at 3.1-? resolution, which we compare with a structure of the VO region with Vph1p that we determine to 3.2-? resolution. These maps reveal differences in the surface charge near the cytoplasmic proton half-channel. Both maps also show the presence of bound lipids, as well as regularly spaced densities that may correspond to ergosterol or bound detergent, around the c-ring.
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Author contributions: T.V., C.V.R., and J.L.R. designed research; T.V., D.W., and V.B.-E. performed research; T.V. and S.A.B. contributed new reagents/analytic tools; T.V., D.W., V.B.-E., and J.L.R. analyzed data; and T.V. and J.L.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: EM maps have been deposited in the Electron Microscopy Data Bank (EMDB), https://www.ebi.ac.uk/pdbe/emdb/ (accession nos. EMD-0644–EMD-0648); coordinates have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6O7T–6O7X).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1814818116/-/DCSupplemental.
Published under the PNAS license.