FIGURE 3:

Components of the mitochondrial PQC system are not affected by the accumulation of aggregated proteins. (A) Coaggregation of mitochondrial PQC system components. WT yeast cells expressing reporter proteins for 24 h were lysed and centrifuged at 20,000 × g. Total cell extracts (T), supernatants (S), and pellet fractions (P; 3 × amounts) were analyzed by Western blot using antibodies against GFP and the indicated mitochondrial PQC components. (B) Long-term stability of IMiQ aggregates. Expression of mtGFP-DHFR_ds was blocked after 2.5 h by addition of 2% glucose. Cells were further incubated at 30°C for the indicated times. Aggregates were analyzed by Western blot as described. Aco1 was used as a control protein. (C) Protein levels of mitochondrial PQC components. Cell extracts containing destabilized or control proteins expressed for 5, 10, or 24 h were analyzed by Western blot using the indicated antibodies. The cytosolic protein Pgk1 was used as loading control. (D) Protein levels of mitochondrial PQC components in cells incubated at 30 or 42°C for 4 h were analyzed by Western blot as above. (E) Growth phenotype of mtGFP-DHFR_ds expressing cells. After 10 h expression of indicated reporter proteins, cells were incubated at 30 or 42°C (heat shock) for 1 h and grown on fermentable (2% glucose) or nonfermentable (3% glycerol) medium at 30°C.