Figure 6.

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.