Figure 1. The temperature sensitive proteins sam35-2HA^ts and sen2-1HA^ts are novel thermosensitive substrates for mitochondrial quality control.

(A) Spot growth assay of cells expressing chromosomal SAM35HA, sam35-2HA^ts, SEN2HA, or sen2-1HA^ts (yMM36, 37, 40, and 41, respectively) at permissive (25°) or non-permissive (37°) temperatures. (B) Wild type (WT; WCG4a) yeast were treated with cycloheximide (CHX) at 25°C or 37°C and analyzed at the indicated times to assess the degradation of centromeric (CEN) plasmid-expressed SAM35HA, sam35-2HA^ts, SEN2HA, or sen2-1HA^ts (pMM158, 157, 159, 160, respectively). The ts- proteins were detected by immunoblotting with HA antibody. Phosphoglycerate kinase (PGK) served as a protein loading control. Graphed below is the mean and standard deviation (SD) of the PGK-normalized HA signal at each time point for three biological replicates. (C) Live-cell microscopy analysis of agarose-embedded WT cells (WCG4a) co-expressing a mitochondrial-matrix targeted RFP (mtRFP; pMD12) and either sam35-2GFP^ts (pMD1) or sen2-1GFP^ts (pMD4) at the indicated times after temperature shift to 37°C. CHX was also added at 0 min, although CHX diffusion through agarose is likely problematic. ‘Merge’ of GFP (green) and RFP (magenta) channels and differential interference contrast (DIC) are shown; Scale bar = 10 μm. (D) Lysates of spheroplasted yeast from the strains used in B were fractionated at 12,000xg at 37°C into mitochondrial pellet (P) and post-mitochondrial supernatant (S). Fractions were subject to immunoblotting with antibodies to HA, PGK (cytosolic protein control), and PORIN (mitochondrial protein control).

Figure 1—figure supplement 1. The temperature sensitive proteins sam35-2HA^ts and sen2-1HA^ts are novel thermosensitive substrates for mitochondrial quality control.

(A) Amino acid substitutions in the sam35-2^ts and sen2-1^ts proteins compared to the WT proteins. (B) Cells with chromosomal SAM35HA, sam35-2HA^ts, SEN2HA, or sen2-1HA^ts (yMM36, 37, 40, and 41, respectively) were treated with cycloheximide (CHX) at 25°C or 37°C and analyzed at the indicated times to assess the degradation of genomic SAM35HA, sam35-2HA^ts, SEN2HA, or sen2-1HA^ts. Ts- proteins were detected by immunoblotting with HA antibody. Phosphoglycerate kinase (PGK) served as a protein loading control. Percentage of each ts- substrate remaining is labeled for each time point under blots. (C) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2GFP^ts or sen2-1GFP^ts (pMD1 or 4, respectively) in WT yeast cells (WCG4a). (D) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2HA^ts or sen2-1HA^ts (pMM157 or 160, respectively) in WT (WCG4a) cells. (E) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2HA^ts or sen2-1HA^ts (pMM157 or 160, respectively) in spheroplasts generated from WT (WCG4a) and pre1-1 pre2-2 (WCG4-11/21a) yeast cells. (F) Lysates from strains expressing genomic sam35-2HA^ts or sen2-1HA^ts (yMM37 and 41, respectively) were fractionated at 12,000xg at 37°C into mitochondrial pellets (P) and post-mitochondrial supernatants (S). Fractions were subject to immunoblotting with antibodies to HA, PGK, and PORIN.