Mitochondrial partitioning affects cytosolic protein aggregate retention. (A) Cells expressing Hsp104Y662A-GFP, mtRFP, and either Myo2-Fis1 from a multicopy plasmid or an empty vector control were grown at 30°C and stained with calcofluor. After applying a heat shock at 42°C for 5 min, cultures were incubated in the absence of calcofluor at 30°C for 2–3 h. Cells with medium-sized buds lacking calcofluor staining were analyzed by fluorescence microscopy. Bar, 5 µm. (B and C) Cells prepared as in A were scored for aggregate retention (i.e., aggregates were only present in the mother cell) or mitochondria-associated aggregates in buds. (D–I) Cells were analyzed as in A–C. Representative images of cells showing no aggregate retention are shown in E. Buds were quantified that showed no aggregate retention (i.e., one or more aggregates were visible in the bud; F) and contained aggregates that were not associated with mitochondria I. In G–I, Δmyo2 cells expressed MYO2 alleles from plasmids. Arrowheads indicate bud-localized aggregates that are not associated with mitochondria. Data pooling and statistics are detailed in Table S4.
