Figure 4.

Import of denatured Tom40 precursor. (A) Denaturation of Tom40 precursor reduces the insertion efficiency in reversible manner. Radiolabeled native Tom40 precursor or urea-treated precursor (3 μl each) was added to either 50 μg mitochondria in 100 μl import buffer containing 10% glycerol (3 μl reticulocyte lysate/100 μl reaction volume), or to a mixture of 60 μl import buffer (containing 10% glycerol) with 30 μl reticulocyte lysate for 20 min at 0°C, followed by the addition of 50 μg mitochondria (33 μl reticulocyte lysate/100 μl reaction volume). In both cases samples were incubated with mitochondria for 20 min at 15°C followed by reisolation by centrifugation, resuspension in import buffer, and dividing the samples into two aliquots. One was left on ice while the other was treated with PK. (B) Denaturation with urea has an opposite effect on import of Tom40 as compared with import of pSu9(1-45)-DHFR. Radiolabeled native or urea-denatured Tom40 and pSu9(1-45)-DHFR precursors were coincubated with 50 μg mitochondria at 15°C. After the indicated times, PK was added followed by centrifugation of the mitochondria and analysis of the proteins by SDS-PAGE (left panel). For comparison and for identification of the bands observed in the left panel, the native precursors were imported separately into mitochondria at 25°C and half of each reaction was treated with PK (right panel). The bands from the coimport experiment corresponding to the 26-kD fragment (F-26) of Tom40 and to the mSu9(1-45)-DHFR were quantified and presented as a percentage of the input (lower panel).