Spatiotemporal organization of protein aggregates in yeast cells. (A) Time-dependent changes in the localization of mCherry-VHL, Hsp104-mCFP, and mCitrine-luciferase (all green) in WT cells grown at 30°C and after a shift to 37°C for 30 and 180 min. MG132 was added before the temperature shift. Nuclei were visualized by coexpressing HTB1-cerulean or HTB1-mCherry (red). (B) Number (shaded bars) and localization (colored bars) of mCherry-VHL, Hsp104-mCFP, and mCitrine-luciferase aggregates in WT cells after incubation at 37°C for 30 and 180 min. The total number of foci per cell (juxtanuclear and peripheral) is depicted in all diagrams on the x axis. Quantifications are based on the analysis of 50–120 cells and are representatives of multiple experimental repeats. (C) Different substrates are sorted to the same compartments. mCherry-VHL (red) and mCitrine-luciferase (green) were coexpressed in WT cells. Protein localizations were determined after a temperature shift to 37°C for 180 min, revealing colocalization of mCherry-VHL and mCitrine-luciferase. Nuclei were visualized by coexpressing HTB1-cerulean (blue). The dashed lines indicate the border of respective yeast cells. Bars, 1 µm.