Overexpression of SidK affects lysosome positioning and pH. (A) Localization of endogenous LAMP1 (magenta) in untreated cells. (B) Localization of LAMP1 (magenta) in cells expressing SidK-mCherry (green). Side panels show individual SidK and LAMP1 channels in area denoted by dotted square, 2.0× magnification. Arrowheads mark sites of peripheral LAMP1 accumulation. (C) Localization of LAMP1 (magenta) in cells treated with 250 nM concanamycin A for 1 h. Arrowheads mark sites of peripheral LAMP1 accumulation. (D) Retention of cresyl violet (magenta) in cells expressing SidK-GFP (green) with lysosomes preloaded with Alexa Fluor 647–conjugated dextran (dex; blue). Side panels show individual channels of area denoted by dotted square, 2.7× magnification. (E) Cells expressing SidK-GFP (green) and labeled for lysosomes (blue) were treated with 250 nM concanamycin A and 10 mM NH₄Cl for 30 min to neutralize luminal pH. After treatment, cells were incubated with cresyl violet as in D. Side panels: 2.7× magnification. (A–E) Extended focus images representative of ≥30 fields from three or more experiments of each type. All scale bars: 5 µm. (F) Lysosomal pH determined in SidK^– and SidK⁺ HeLa cells. Lysosomes of control or SidK-mCherry–transfected HeLa cells were loaded overnight with FITC-dextran. pH was measured by ratiometric fluorescence microscopy. For each condition, three independent experiments were quantified, with ≥20 cells per replicate. Data are mean ± SEM. P calculated using unpaired, two-tailed Student’s t test. (G) Lysosomal V-ATPase activity was measured in control and SidK-mCherry–expressing HeLa cells acutely treated with 500 nM concanamycin A. V-ATPase activity was determined from the inverse rate of alkalization upon addition of concanamycin A. For each condition, three independent experiments were quantified, with ≥10 cells per replicate. Data are mean ± SEM. P calculated using unpaired, two-tailed Student’s t test.