FIGURE 5.

Analysis of TOM complex composition and function in mitochondria from selected Tom40 Cys-mutant strains. A, cells from the indicated strains (top of panel) were grown in liquid culture, and mitochondria were isolated. Mitochondrial proteins were subjected to SDS-PAGE followed by transfer to nitrocellulose, and immunodecoration with the antibodies indicated on the left. The control strain was 76-26, which contains wild type Tom40. B, isolated mitochondria from the strains indicated (top of panel) were dissolved in 1% digitonin or 1% n-dodecyl-β-d-maltoside (DDM) for analysis of complex stability using BNGE. Blots were decorated with Tom40 antibody. The controls are Tom6 RIP (a strain lacking Tom6 that shows severely reduced TOM complex stability (51)) and 76-26 (control), which contains wild type TOM complex. C, radiolabeled matrix precursor F₁β and inner membrane precursor ADP/ATP carrier (AAC) were incubated (for 5 or 20 min, as indicated) with mitochondria isolated from the Tom40 Cys-substitution strains or the 76-26 control. Following import, mitochondria were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membrane, and import was analyzed by autoradiography. Lysate represents 33% of the radiolabeled lysate added to each import reaction. D, radiolabeled Tom40 precursor was incubated for 5 and 20 min with mitochondria isolated from the Tom40 Cys-substitution strains indicated (top of panel) as well as the 76-26 control strain. Mitochondria were dissolved in 1% digitonin and subjected to BNGE. The proteins were transferred to PVDF membrane and analyzed by autoradiography. The size of the mature TOM complex (400 kDa), and assembly intermediates I (250 kDa) and II (100 kDa) are indicated on the left. * indicates an undefined band.