FIG. 1.

Stability of the 400K GIP complex. (A) Mitochondria (100 μg of protein) were lysed in solubilization buffer containing 1% digitonin and the indicated concentration of urea for 10 min on ice. After a clarifying spin, protein complexes were separated by BN-PAGE and blotted onto PVDF membranes, followed by immunodecoration using antisera against Tom40, Tom22, and porin. As control, 25% of the samples were directly subjected to SDS-PAGE and immunodecoration. (B) Mitochondria (100 μg of protein) were resuspended in 100 mM Na₂CO₃ (pH 11.5) and incubated for 30 min on ice. After centrifugation at 100,000 × g, the nonextractable pellet was lysed in digitonin buffer and subjected to BN-PAGE, followed by immunodecoration against Tom40 and Tom22 (lane 1); the control represents mitochondria without carbonate treatment. A control sample, the nonextractable pellet, and the TCA-precipitated supernatant (Sn.) were also analyzed by SDS-PAGE, showing the correct fractionation patterns (lanes 6 to 8). BN-PAGE of control samples and the carbonate-resistant fractions, followed by Western blot analysis using antisera against Tim22, Tim23, the β subunit of the F₁-ATPase, and Hsp60, revealed no carbonate-resistant complexes (lanes 9 to 12). For salt extraction (lanes 3 to 5), mitochondria were resuspended and incubated in SEM buffer containing the indicated NaCl concentrations for 10 min on ice. After the salt was washed off with SEM buffer, the mitochondria were lysed in digitonin-containing buffer and subjected to BN-PAGE.