Figure 1.

Purification and reconstitution strategy. (A) Yeast cells are lysed and vacuoles purified by flotation on a Ficoll gradient. (B) MSP with an N‐terminal extension containing a BAP tag for in vivo biotinylation and a Prescission protease cleavage site (PPase) is expressed in E. coli. Vacuoles are detergent solubilized and total vacuolar membrane proteins are nanodisc‐reconstituted using vacuolar lipids and biotinylated MSP. From this mixture of nanodisc‐reconstituted membrane proteins, V‐ATPase‐containing nanodiscs are purified by affinity chromatography using a FLAG‐tag at the G subunit N‐terminus. (C) V‐ATPase reconstituted in biotinylated and vacuolar lipid containing nanodiscs is schematically depicted as V ₁ V ₀ND. V‐ATPase is composed of a cytosolic V ₁‐ATPase sector and a membrane integral V ₀ proton channel sector.