Figure 4.

VopE targeted to yeast mitochondria and does not impair respiration. (A) Plasmids encoding mCherry or VopE-mCherry variants were introduced into yeast reporter strain BY4741-COX4-GFP. Mid-log phase cells were induced for 6 h by the addition of galactose. Mitochondria were visualized by COX4-GFP (targeting to mitochondrial matrix). Cells were examined by confocal laser scanning microscopy to monitor the localization patterns of VopE-mCherry constructs. Scale bar equals to 5 μm. (B) Detection of VopE-3XFLAG in mitochondrial fraction and whole cell lysate of 6 h galactose induced cultures with anti-FLAG antibody. Mitochondrial fraction was confirmed with anti-porin antibody. (C) BY4741 transformants expressing VopE either constitutive (pAMU10, low copy vector, top panel) or inducible (pESCLEU, high copy vector, bottom panel) were subjected to spotting on selective solid media containing glucose or 2% glycerol. For liquid growth induction, liquid cultures were pretreated with galactose for 6 h before spotting.