FIGURE 10.

Cytosolic Grx3-Grx4 affects Aft1 function. A, cells expressing Fis1-Grx4-c-Myc fusion protein were grown to midlog and processed for immunofluorescence using rabbit anti-c-Myc (1:100; Covance) and mouse anti-porin (1:100; Invitrogen), followed by Alexa 594-conjugated goat anti-rabbit IgG and Alexa 488-conjugated goat anti-mouse IgG (1:750; Invitrogen). The c-Myc-tagged Fis1-Grx4 co-localized with the mitochondrial outer membrane protein porin, showing that the fusion protein is tethered to the mitochondria, thus restricting Grx4 to the cytosol. B, wild type (WT), Δgrx3Δgrx4, or Δgrx3Δgrx4 transformed with empty vector, pGRX4-c-Myc, or pFIS1-GRX4-c-Myc were grown to midlog, and samples were harvested for FET3 and CMD1 S1 analysis or Western blot using an anti-c-Myc tag antibody. Grx4-c-Myc and Fis1-Grx4-c-Myc are expressed at comparable levels.