Fig. 2. Pos5p is a mitochondrial matrix protein that is needed for proper mitochondrial function. (A) The indicated strains were tested for growth by plating cell dilutions as in Figure onto YPD (–O2, +air, +100% O2) or YPG (glycerol) plates in air. –O2, +air, +100% O2 = same as in Figure . Strains utilized: wild-type, BY4741; pos5Δ, CO205; pos5Δ + vector, strain CO205 transformed with vector pAA1; pos5Δ + POS5-GFP, strain CO205 transformed with plasmid pCO101. (B) Strain BY4741 transformed with pCO101 (Pos5–GFP) or pAA1 (control) was prepared for fluorescence microscopy as described in Materials and methods. Merged = merged images of DAPI and GFP fluorescence. (C) Cell lysates were prepared from strain BY4741 transformed with the Pos5–GFP plasmid pCO101 (P) or the vector control pAA1 (V), and 140 µg of total cell protein was fractionated into post-mitochondrial supernatant (PMS) and crude mitochondria (Mito) (left). Where indicated, mitochondria (25 µg of protein) were fractionated further into intermembrane space (IMS) and matrix components (right). All samples were subject to SDS–PAGE and immunoblotting using antibodies directed against either GFP (marking Pos5p), cytochrome b2 (mitochondrial IMS) or Mas2 (mitochondrial matrix). (D) The indicated strains were tested for growth as in (A). Strains utilized: wild-type, BY4741; pos5Δ, BY4741 pos5Δ::kanMX4; coq1Δ, CO217; pos5Δ coq1Δ, CO200.