Fig. 1. Import of radiolabelled DIC into isolated mitochondria. (A) The DIC is a protein of 298 residues containing six putative membrane-spanning domains (grey segments). A carrier signature is found in two positions (CS1 and CS2). (B) Wild-type DIC and a mutant version lacking the carrier signatures (exchange of the corresponding residues with alanine, CS1/2) were synthesized in reticulocyte lysate in the presence of ³⁵S-labelled methionine and centrifuged at 2°C for 45 minutes at 100,000 g. The pellets (P) and samples of the supernatants (S) were analyzed by SDS-PAGE and fluorography to determine the distribution of the radiolabelled DIC or DIC_CS1/2, respectively. Four samples were tested in parallel to calculate the standard deviation. (C) Mitochondrial import of DIC and derivatives lacking carrier signature CS1, CS2 or both. The ³⁵S-labelled proteins were incubated with isolated yeast mitochondria at 25°C for different times, as indicated. The mitochondria were subsequently treated with proteinase K, re-isolated and the proteins were separated by SDS-PAGE. A PhosphorImager was used for quantification; the highest value was set to 100% (control). (D) The radiolabelled proteins were imported into mitochondria for 5 minutes and analyzed as in C. Standard samples of the reticulocyte lysates were included to allow the calculation of the import efficiency (n=3). (E) Import of DIC and derivatives containing the amino acid exchanges P33A, D35A or K38A. The radiolabelled proteins were imported into isolated mitochondria for different times and subsequently analyzed as in C. Bars represent standard deviation.