Figure 2
BamA and Tob55 are not interchangeable.
(a) Plasmids encoding Tob55, BamA, or the various Tob55/BamA hybrids were transformed into a strain expressing Tob55 under the control of an inducible GAL10 promoter. Cells were grown first on galactose and then were shifted to glucose-containing medium. After 48 h on glucose, all cells were diluted to the same OD600 and their growth was further monitored at the indicated time periods. (b) The cells described in (a) were grown initially for 30 h on galactose-containing medium and then were analyzed at the indicated temperatures by drop-dilution assay on synthetic (SD-Trp) or rich (YPD) glucose-containing medium. (c) Cells as in (a) were grown first on galactose-containing medium and then were shifted to glucose-containing medium. Cells were harvested directly before the shift (t = 0) or 30 h afterwards (t = 30 h). Proteins were analysed by SDS-PAGE and immunodetection with antibodies against the indicated proteins or the HA-tag. The Tob55-BamA variants are indicated with an asterisk (*) whereas a non-specific band of the Tob55 antibody is depicted with a hash (#). (d) Mitochondria were isolated from the Tob55-depleted cells expressing plasmid-encoded Tob55 (variant A), hybrid D, or variant G. The organelles were solubilized with 1% digitonin and were analysed by BN-PAGE and immunodecoration with antibody against Tob55. The bands corresponding to the TOB complex and Tob55 monomeric forms are indicated. (e) Mitochondria (Total, T) described in part (d) were subjected to alkaline extraction separating membrane-embedded proteins in the pellet (P) from soluble proteins in the supernatant (S). Proteins were analysed by SDS-PAGE and immunodecoration with antibodies against Tob55, Tom20, or Hep1 (a soluble protein in the mitochondrial matrix). For clarity, a letter code of the relevant variants and their scheme are shown.
