a–e, Representative images and quantification of localization of HA–TBC1D15 to mitochondria (stained with endogenous TOM20) in fixed HeLa cells showing that mitochondrial localization is not disrupted by TBC1D15 GAP mutants (D397A or R400K) but is disrupted by mutating the FIS1-binding site of TBC1D15 (Δ231–240) (n = 293 cells, WT; n = 228 cells, D397A; n = 181 cells, R400K; n = 379 cells, Δ231–240). Δ231–240 versus WT (*P = 0.0178), D397A (*P = 0.0131), and R400K (*P = 0.0112), ANOVA with Tukey’s post-hoc test. f, Quantification showing that localization of YFP–TBC1D15 to mitochondria is greatly decreased by the Flag–FIS1(LA) mutant (which cannot bind TBC1D15) as compared to wild-type Flag–FIS1 (n = 290 cells, FIS1; n = 281 cells, FIS1(LA)). ***P < 0.0001, unpaired two-tailed t-test. g, Examples of HA–TBC1D15 GAP mutants (D397A and R400K) or FIS1-binding mutant (Δ231–240) inducing enlarged lysosomes (white arrows) (LAMP1–mGFP) not observed in cells expressing wild-type HA–TBC1D15 (n = 293 cells, WT; n = 228 cells, D397A; n = 181 cells, R400K; n = 379 cells, Δ231–240). Data are means ± s.e.m. Scale bars, 10 μm (a–d, g); 1 μm (a–d, insets).