FIG. 3.

Relation of Tom20 and Tom70 to the yeast GIP complex. (A) BN-PAGE of mitochondria (50 μg of protein) lysed in different concentrations of digitonin, followed by immunodecoration with Tom40 antiserum. The high-molecular-weight complex observed at 0.1% digitonin is indicated by an asterisk. (B) Two-dimensional electrophoresis, BN-PAGE followed by SDS-PAGE, of mitochondria (100 μg of protein) solubilized in 1% digitonin (top) or 0.1% digitonin (bottom). As a control, mitochondria (20 μg of protein) were loaded directly onto the SDS-PAGE and electrophoresed in one dimension (bottom, Mito control). Immunodecoration was performed with antiserum directed against Tom70, Tom40, Tom20, or Tom5. (C) Binding of Tom proteins from Tom22-His mitochondria (left) or wild-type mitochondria (right) onto Ni-NTA. Mitochondrial membranes were isolated and solubilized as described in Materials and Methods. After binding and collection of the flowthrough fraction, the column was washed with 50 mM imidazole followed by 80 mM imidazole, and the TOM complex was eluted at 200 mM imidazole. Aliquots of each fraction were subjected to SDS-PAGE and immunoblotting.