FIG. 3.

Effect of anti-hTom22 antibodies on import of preproteins into isolated mitochondria. (A) Isolated COS-7 cell mitochondria (25 μg) were preincubated in the presence of 6 μg of nonimmune IgG (NI) or affinity-purified anti-T22N antibody (22N) for 20 min at 25°C in the import reaction mixture (50 μl). ³⁵S-labeled reticulocyte lysate translation product containing human pOTC (10 μl) was then added to the mixture and incubated at 25°C. The import reaction was stopped at the indicated times and subjected to SDS–10% PAGE. The radioactive polypeptides were visualized by image plate analysis (left panels), and the radioactive mature OTC on the SDS-polyacrylamide gels was quantified (right panel). “20%” represents 20% of input pOTC. The percent import represents the amount of mature OTC compared with the input pOTC. (B) Import of ³⁵S-labeled human pOTC was performed in the absence (control) or presence of indicated amounts of nonimmune IgG (NI) or affinity-purified anti-T22N (22N) or anti-T22C (22C) antibodies for 16 min at 25°C and was analyzed as described for panel A. Import without antibody was set as 100%. (C) Import of human pOTC (hpOTC), pOTC-GFP, rat pOTC (rpOTC), pre-serine:pyruvate aminotransferase (pSPT), and pre-aspartate aminotransferase (pAAT) was performed in the presence of 6 μg of nonimmune IgG (NI) or affinity-purified anti-T22N antibody (22N) for 16 min at 25°C and was analyzed as described for panel A. The percent import represents the amount of mature proteins compared with the input precursors. Values are represented by means ± standard deviations of three independent experiments. p and m, precursor and mature forms, respectively.