WASH causes recycling of the V-ATPase. (a) Confocal imaging of wshA⁻ cells expressing GFP-WASH and VatB-mRFP after endocytosis of 0.5-µm agarose beads. Frames are taken from Video 3. Insets show a magnified view of the vesicle on which WASH is acting. (b) Quantification of various vesicular proteins during postlysosome formation. Graphs show that the loss of V-ATPase immediately follows the arrival of WASH and coronin, whereas the previously described (Maniak, 2001) postlysosome marker vacuolin is present much earlier than neutralization and rises steadily. The representative curves are from an experiment that was performed at least five times. (c) Rapid widefield oblique illumination imaging of small vesicles containing both GFP-WASH and VatB-mRFP budding a single lysosome. Frames are taken from Video 4. Bar, 1 µm.