Analysis of endosomal and vacuolar markers relative to Ivy1 in HOPS mutant cells. (A) Localization of Ivy1 relative to Vps8, Vps4, Apl5, and Fab1 (quantification is shown in Fig. 5, A and C). vps11-1 ts cells expressing mGFP-tagged Ivy1 and mCherry-tagged Vps8, Vps4, and Apl5 or mScarlet-tagged Ivy1 and mNeon-tagged Fab1 were grown at 24°C in a synthetic medium, and then shifted or not to 37°C for 1 h. Vacuoles were stained with CMAC. The cells were analyzed by fluorescence microscopy, and individual slices are shown. Arrows show colocalizing dots. (B) Localization of Ivy1 relative to Vps39, Vps41, Ccz1, and the artificial cargo GNS. vps11-1 cells expressing mScarlet-tagged Ivy1; mNeon-tagged Vps39, Vps41, or Ccz1; or GFP-tagged GNS were grown and analyzed as in A. Scale bar, 5 µm. Arrows show colocalizing dots. (C) Quantification of Ivy1 colocalizing with Vps39, Vps41, and Ccz1 puncta. Cells (n ≥ 200), Ivy1 dots (n ≥ 250), Vps39 dots (n ≥ 100), Vps41 dots (n ≥ 100), or Ccz1 dots (n ≥ 200) were quantified by ImageJ. Error bars correspond to SD of three independent experiments. ns, P > 0.05; *, P ≤ 0.05 (Student’s t test).