SidK interacts with the V-ATPase in vitro and when expressed in mammalian cells. (A) SDS-PAGE of 3×FLAG SidK_1-278 illustrates purity of the probe and its ability to allow isolation of yeast V-ATPases by affinity chromatography. Bands corresponding to SidK_1-278 and individual subunits of V-ATPase are labeled. (B) Model of yeast V-ATPase with SidK_1-278-GFP bound, generated from PDB accession nos. 5VOX, 1GFL, and 6O7T. (C) Visualization of SidK_high cells. HeLa cells were transfected with SidK-GFP (green) and lysosomes loaded with Alexa Fluor 647–labeled dextran (magenta). Side panels show individual SidK and dextran channels in dotted square, 2.2× magnification. (D) Visualization of SidK_low cells (green) labeled as in C. Side panels show individual SidK and dextran channels, 2.0× magnification. (E and F) Lysosomes identified by LAMP1 immunostaining (magenta) after expression of high levels of SidK-GFP (SidK_high; green). Prior to fixation and immunostaining, cells were left untreated (E) or permeabilized with PLY to remove excess cytosolic SidK-GFP (F). Side panels show individual SidK and LAMP1 channels. Here and elsewhere, cell outlines are indicated by dotted lines. Images in C-F are compressions of confocal stacks representative of ≥30 fields from three or more experiments of each type. Scale bars: 5 µm.