Figure 1.
Purification and characterization of VoND. A and B, schematic of V-ATPase regulation by reversible disassembly. C, purification strategy. Yeast vacuoles are isolated by flotation on a Ficoll gradient (panel i). Detergent-solubilized vacuolar proteins are mixed with biotinylated MSP (panel ii) and reconstituted into lipid nanodiscs followed by α-FLAG affinity capture of VoND (panel iii). D, size-exclusion chromatography of VoND. E, peak fractions were resolved using SDS-PAGE. F, negative stain EM of purified VoND. Bar, 20 nm.