Browsing Upstate Medical University by Subject "LIPID"
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NEAR-ATOMIC RESOLUTION STRUCTURE OF THE YEAST VACUOLAR (V-) ATPASE MEMBRANE SECTOR Vo IN LIPID NANODISCVacuolar ATPase (V-ATPase) is a large multisubunit enzyme that acidifies subcellular organelles and the extracellular space. Its activity is regulated by reversible disassembly, causing V-ATPase dissociation into soluble V1-ATPase and membrane-integral Voproton channel sectors.The goal of this thesis project was to observethe yeast Voin a physiologically relevant, auto-inhibited state, i.e. in its form dissociated from the ATPase sector, V1, in order to better visualizethe closed pore and to identify testable hypotheses on why the pore remains closed following dissociation from V1. Towards this aim we present two chapters:In Chapter 1, we detail a single-particle negative stain EM study of lipid nanodisc reconstituted Vo,which suggesteddissociated Vois halted in theso-called rotational state 3 of the holo-enzyme.We performed site directed mutagenesis and binding studies of subunits aand dto test and validate this hypothesis.In Chapter 2,we further detaillipid nanodisc reconstitutedVoin a high-resolutioncryoEM structure,confirmingour earlier identification of Voresting in rotational state 3, andproviding structural information of the sectorat the amino acid level. Through this work we proposed apossible mechanism for transmembrane proton transport in the V-ATPaseandidentifieda new subunit member of Vo, assembly factor Voa1.The studies shown here highlight the potential of lipid nanodisc reconstitution of membrane protein complexes, give insight into a conformational mismatch between autoinhibited V1and halted Vowith the implication that the mismatch may serve to prevent unintended reassembly of V-ATPase upon activity silencing, and propose a chemical basis for transmembrane proton transport in the Voproton pore.