Skip to main content
. 2017 Feb 22;45(6):3323–3340. doi: 10.1093/nar/gkx127

Figure 1.

Figure 1.

Δppr10 cells exhibit a range of phenotypes indicative of defective mitochondrial respiration. (A) Growth defects of Δppr10 cells on various media. WT and Δppr10 cells were grown in YES medium to stationary phase and 10-fold serial dilutions were spotted onto rich media containing 3% glucose (YES+Glu), 6% glycerol (YES+Gly) or 4% galactose and 0.1% glucose (YES+Gal). Plates were incubated at 30°C for 6–7 days. (B) Δppr10 cells are highly sensitive to antimycin A. Stationary-phase WT, Δppr10, Δppr2 and Δppr6 cells were spotted in 10-fold serial dilution onto YES plates in the absence or presence of 100 μg/ml antimycin A and grown at 30°C for 2–3 days. (C) The PPR motifs of S. pombe Ppr10 are required for respiratory growth. A 10-fold dilution series of WT cells carrying empty plasmid pREP82X (WT/pREP82X), or Δppr10 cells carrying pREP82X-Ppr10 expressing full-length Ppr10 (Δppr10/Ppr10), pREP82X-Ppr10ΔPPR expressing Ppr10 lacking two predicted PPR motifs (Δppr10/Ppr10ΔPPR) or pREP82X (Δppr10/pREP82X) was spotted onto YES or YNB plates containing 3% glucose (YNB+Glu), 6% glycerol (YNB+Gly) or 4% galactose and 0.1% glucose (YNB + Gal), and grown at 30°C. (D) A western blot shows the expression of full-length Ppr10 and a mutant lacking the PPR motifs. Extracts were prepared from Δppr10 cells carrying pREP82X, or a plasmid either expressing 5FLAG-tagged full-length Ppr10 (Ppr10-FLAG) or mutant Ppr10 lacking two predicted PPR motifs (Ppr10ΔPPR-FLAG). Mitochondrial heat shock protein Hsp60 detected by anti-human mitochondrial matrix protein HSP60 Ab serves as a loading control. (E and F) Deletion of ppr10 results in a rapid loss of cell viability. Cells were grown overnight in YES, diluted to an initial OD600 of 0.2 and grown for 84 h. At 12 h intervals, aliquots of the cultures were 10-fold serially diluted, and either spotted or plated onto YES plates. Plates were either photographed (E) or grown colonies counted to determine cell viability (F). The percentage of cell viability was calculated by normalization to the viability of cells at 12 h.