Analysis of proteins found on Vps4-positive LEs in HOPS mutant cells. (A) Localization of the MVB marker Vps4 relative to endosomal proteins. vps11-1 ts cells expressing mGFP-tagged Vps4 and mCherry-tagged Vps8, Vps21, and Pep12 or mCherry-tagged Vps4, and mGFP-tagged Ypt7 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 are shown via individual slices. Scale bar, 5 µm. Quantification is shown in Fig. 5 D. Arrows show colocalizing dots. (B) Localization of Vps4 relative to Vps39, Vps41, Ccz1, Fab1, and Apl5. vps11-1 cells expressing mCherry-tagged Vps4 and mNeon-tagged Vps39, Vps41, Ccz1, and Fab1 or mGFP-tagged Vps4 and 3xmCherry-tagged Apl5 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. Cells were analyzed by fluorescence microscopy, and individual slices are shown. Scale bar, 5 µm. Arrows show colocalizing dots. (C) Quantification. Cells (n ≥ 150), Vps4 dots (n ≥ 400), Vps39 dots (n ≥ 100), Vps41 dots (n ≥ 100), Ccz1 dots (n ≥ 200), or Fab1 dots (n ≥ 50) were quantified by ImageJ. Error bars represent SD of three independent experiments. *, P ≤ 0.05; ***, P ≤ 0.001 (Student’s t test).