Figure 3.
SE identity depends on MVB functionality. (A and B) Localization of Ivy1 relative to endosomal proteins in ESCRT mutants. Wild-type or vps4Δ cells expressing mGFP-tagged Ivy1 and mCherry-tagged Vps8, Vps21, Ypt7, and Kog1 or expressing mCherry-tagged Ivy1, GFP-tagged Ego1, and mNeon-tagged Fab1 were grown in a synthetic medium. Vacuoles were stained with CMAC. The cells were analyzed by fluorescence microscopy and are shown as individual slices. Scale bar, 5 µm. Arrows show colocalizing dots. (C) 3D track of the mean fluorescence intensity of Ivy1-mGFP from Videos 3 and 4. Wild-type or vps4Δ cells expressing mGFP-tagged Ivy1 were grown in a synthetic medium. Vacuoles were stained with CMAC, and the data were analyzed as in Fig. 2 F. (D) Quantification related to A and B. Cells (n ≥ 200), Ivy1 dots (n ≥ 200), Vps4 dots (n ≥ 400), Vps21 dots (n ≥ 300), Ypt7 dots (n ≥ 100), Ego1 dots (n ≥ 50), Kog1 dots (n ≥ 100), or Fab1 dots (n ≥ 50) were quantified by ImageJ. Error bars represent SD of three independent experiments. ns, P > 0.05 (Student’s t test). (E) Localization of Tor1 or Ego1 relative to Ivy1 and Cps1 in vps4∆ cells. Cells expressing mGFP-tagged Tor1 or Ego1 with HaloTag-Ivy1 and mCherry-Cps1 were grown in a synthetic medium. The cells were incubated with the Janelia fluor 646 HaloTag ligand for 1 h and washed eight times before imaging. The cells were analyzed by fluorescence microscopy and are shown as individual slices. Scale bar, 5 µm. Arrows show colocalizing dots. (F) Quantification of Tor1 or Ego1 dots colocalizing with Ivy1 and/or Cps1 from E. Cells (n ≥ 200), Tor1 dots (n ≥ 100), Ego1 dots (n ≥ 100), Ivy1 dots (n ≥ 200), and Cps1 dots (n ≥ 200) from three independent experiments were quantified by ImageJ. (G) Schematic model showing the different populations of mGFP-Tor1 and Ego1-mGFP dots analyzed in F. Dark gray spots indicate Tor1 or Ego1 dots colocalizing with both Ivy1 and Cps1. Blue and red spots indicate Tor1 or Ego1 dots colocalizing with just Ivy1 or just Cps1, respectively. Light gray spots indicate Tor1 or Ego1 dots that show colocalization with neither Ivy1 nor Cps1.

SE identity depends on MVB functionality. (A and B) Localization of Ivy1 relative to endosomal proteins in ESCRT mutants. Wild-type or vps4Δ cells expressing mGFP-tagged Ivy1 and mCherry-tagged Vps8, Vps21, Ypt7, and Kog1 or expressing mCherry-tagged Ivy1, GFP-tagged Ego1, and mNeon-tagged Fab1 were grown in a synthetic medium. Vacuoles were stained with CMAC. The cells were analyzed by fluorescence microscopy and are shown as individual slices. Scale bar, 5 µm. Arrows show colocalizing dots. (C) 3D track of the mean fluorescence intensity of Ivy1-mGFP from Videos 3 and 4. Wild-type or vps4Δ cells expressing mGFP-tagged Ivy1 were grown in a synthetic medium. Vacuoles were stained with CMAC, and the data were analyzed as in Fig. 2 F. (D) Quantification related to A and B. Cells (n ≥ 200), Ivy1 dots (n ≥ 200), Vps4 dots (n ≥ 400), Vps21 dots (n ≥ 300), Ypt7 dots (n ≥ 100), Ego1 dots (n ≥ 50), Kog1 dots (n ≥ 100), or Fab1 dots (n ≥ 50) were quantified by ImageJ. Error bars represent SD of three independent experiments. ns, P > 0.05 (Student’s t test). (E) Localization of Tor1 or Ego1 relative to Ivy1 and Cps1 in vps4∆ cells. Cells expressing mGFP-tagged Tor1 or Ego1 with HaloTag-Ivy1 and mCherry-Cps1 were grown in a synthetic medium. The cells were incubated with the Janelia fluor 646 HaloTag ligand for 1 h and washed eight times before imaging. The cells were analyzed by fluorescence microscopy and are shown as individual slices. Scale bar, 5 µm. Arrows show colocalizing dots. (F) Quantification of Tor1 or Ego1 dots colocalizing with Ivy1 and/or Cps1 from E. Cells (n ≥ 200), Tor1 dots (n ≥ 100), Ego1 dots (n ≥ 100), Ivy1 dots (n ≥ 200), and Cps1 dots (n ≥ 200) from three independent experiments were quantified by ImageJ. (G) Schematic model showing the different populations of mGFP-Tor1 and Ego1-mGFP dots analyzed in F. Dark gray spots indicate Tor1 or Ego1 dots colocalizing with both Ivy1 and Cps1. Blue and red spots indicate Tor1 or Ego1 dots colocalizing with just Ivy1 or just Cps1, respectively. Light gray spots indicate Tor1 or Ego1 dots that show colocalization with neither Ivy1 nor Cps1.

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