Figure 5
PhoE and OmpA have variable dependencies on chaperones.
(a) Crude mitochondria were isolated from Tob55-depleted cells co-expressing OmpA and either plasmid-encoded Tob55 or the indicated variants. In addition, these cells were transformed with an empty plasmid (Ø) or a plasmid encoding mitochondrially-targeted SurA or Skp. Proteins were analyzed by SDS-PAGE and immunodetection with the indicated antibodies. (b) The bands of at least three independent experiments as those described in (a) were quantified and their means ± s.d. are presented. To allow comparison among the various samples, the levels of Tom20 were taken as internal reference. The intensities of the bands from cells transformed with native Tob55 (construct A) in the absence of chaperone were set to 100%. (c,d) Crude mitochondria were isolated from Tob55-depleted cells co-expressing PhoE, the indicated Tob55 variant, and either SurA or Skp. Further treatment and analysis were as described in parts (a) and (b). (e) Mitochondria (Total, T) were isolated from cells co-expressing plasmid-encoded native Tob55 (construct A) and PhoE in the presence or absence of Skp. The organelles were subjected to alkaline extraction separating membrane-embedded proteins in the pellet (P) from soluble proteins in the supernatant (S). Proteins were analyzed by SDS-PAGE and immunodecoration with antibodies against PhoE, Skp, Aco2 (a soluble protein in the mitochondrial matrix), and Tob55. A schematic representation of the relevant Tob55 variants is included.
