Fig. 4. Effect of antibodies against rTOM70 on mitochondrial preprotein import.

(A) Mitochondria were preincubated with the indicated concentrations of preimmune IgG (PI), anti-rTOM70 IgG (70) or anti-rTOM20 IgG (20) at 0°C for 30 minutes, reisolated and subjected to the import of pre-Su9-DHFR, pAd, AAC and rTOM40. When import of AAC or rTOM40 was assayed, the reaction mixtures were treated with proteinase K at 0°C for 30 minutes under hypotonic (for AAC) or isotonic (for rTOM40) conditions after import. After the reaction, 1 mM PMSF was added to the reaction mixtures, the mixture was centrifuged, the precipitates were resolved by SDS-PAGE and the gels were analyzed by FLA2000. Other conditions were as described in the Materials and Methods. The import efficiencies were expressed relative to the control import in the absence of IgG or in the presence of preimmune IgG. Where indicated, the import reactions were performed in the presence of 10 µM CCCP. (B) Effect of anti-rTOM70 IgGs on the MSF- or reticulocyte-lysate-supported mitochondrial import of pAd. ³⁵S-pAd or ¹²⁵I-pAd was subjected to the import reaction in the presence of MSFS (right) or rabbit reticulocyte lysate (left), respectively, using the mitochondria that had been treated with the indicated IgGs (200 µg/ml each) and reisolated. After the reaction, the reaction mixtures were resolved by SDS-PAGE and the gels were analyzed by autoradiography. Other conditions were as described in Materials and Methods.