Schematic diagram of the mitophagy screen and the resulting number of mutants.

Screen for defects in mitophagy. Wild-type (WT; BY4742) and the indicated mutant strains expressing Om45-GFP (top and bottom) were cultured in YPL medium for 12 h and then starved in SD-N for 6 h. The cell lysates equivalent to A₆₀₀ = 0.2 U of cells were subjected to immunoblot analysis with anti-YFP antibody. The position of full-length Om45-GFP and free GFP are indicated.

Om45-GFP processing analysis of novel mutants. Wild-type (WT; TKYM22) and the indicated mutant strains expressing Om45-GFP (top and bottom) were cultured in YPL medium for 12 h and then starved in SD-N for 6 h. The cell lysates equivalent to A₆₀₀ = 0.2 U of cells were subjected to immunoblot analysis with anti-YFP or anti-Pgk1 (loading control) antibodies or antiserum, respectively.

MitoPho8Δ60 analysis of novel mutants. Wild-type (WT; KWY20) and the indicated mutant strains (top and bottom) expressing mitoPho8Δ60 were grown in YPL and shifted to SD-N and SL-N for 4 h. Samples were collected, and protein extracts were assayed for mitoPho8Δ60 activity. The value for the wild-type strain was set to 100%, and the other values were normalized. Values lower than zero are depicted as zero.

Wild-type (WT; WLY176) and the indicated mutant strains expressing Pho8Δ60 were grown in YPD and shifted to SD-N for 4 h. Samples were collected, and protein extracts were assayed for Pho8Δ60 activity. The value for the wild-type strain was set to 100%, and the other values were normalized.

Characterization of Ylr356w. (A) A strain expressing Ylr356w-GFP under the control of the GAL1 promoter (TKYM201) was cultured in YPD medium to midlog phase and shifted to YPGal medium for 4 h. Cells were labeled with the mitochondrial marker MitoFluor Red 589. The localization of GFP and MitoFluor Red were visualized by fluorescence microscopy. DIC, differential interference contrast. Bar, 5 μm. (B) Mitochondria were purified from a strain expressing chromosomally tagged Tim23-myc and Ylr356w-PA as described in Materials and Methods. Equal amounts of the total cell homogenate (T), mitochondrial (M), and supernatant (S) fractions were loaded and detected with antibodies to myc and porin, a purified antibody that recognizes PA, or with antiserum to Pgk1. (C) Isolated mitochondria were treated with proteinase K (PK) with or without hypotonic or Triton X-100 treatment. Samples were TCA-precipitated and subjected to immunoblotting using the appropriate antibodies. (D) Wild-type (WT; TKYM22) and ylr356wΔ strains expressing Om45-GFP were cultured in YPL medium for 12 h and then starved in SD-N for 6 h. The cell lysates equivalent to A₆₀₀ = 0.2 U of cells were subjected to immunoblot analysis with anti-YFP and anti-Pgk1 (loading control) antibody or antiserum, respectively. (E) Wild-type (WT; TKYM22) and ylr356wΔ strains expressing Om45-GFP were cultured in YPL medium for the indicated times. Cell lysates equivalent to A₆₀₀ = 0.2 U of cells were subjected to immunoblot analysis with anti-YFP antibodies and anti-Pgk1 antiserum.