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Fig. 4. Maturation of DIC in the mitochondrial inner membrane. (A) Analysis by BN-PAGE. ³⁵S-labelled DIC and the derivatives ${Delta}$ CS1, ${Delta}$ CS2 and ${Delta}$ CS1/2 were imported into isolated yeast mitochondria for 20 minutes at 25°C, the mitochondria were treated with proteinase K, re-isolated, dissolved in the presence of 1% digitonin, and separated by BN-PAGE. The radiolabelled proteins were visualized using a PhosphorImager. Some samples contained valinomycin to dissipate the mitochondrial membrane potential (– ${Delta}$ ${Psi}$ ). As indicated, mature carrier proteins (import stage V) were usually detected by this method at a range corresponding to 80-120 kDa. (B) 2D analysis of DIC_CS1. A stripe from a BN-PAGE as shown in A was excised and layered on top of an SDS-PAGE for separation of the proteins in a second dimension. The radiolabelled proteins were subsequently visualized using a PhosphorImager. Upper panel, stripe from a DIC_CS1 BN-PAGE sample run in parallel; lower panel, SDS-PAGE. (C) Affinity of DIC for hydroxyapatite. Radiolabelled DIC and DIC_CS1/2 were imported into mitochondria for 15 minutes at 25°C. As indicated, parallel samples contained valinomycin to dissipate the membrane potential (– ${Delta}$ ${Psi}$ ). The mitochondria were then treated with proteinase K, re-isolated and solubilized in the presence of 2.5% Triton X-100. Following a clarifying spin for 5 minutes at 16,000 g, the supernatants were passed through small columns containing hydroxyapatite. The proteins of the eluates were collected by precipitation with trichloro-acetic acid and analyzed by SDS-PAGE and fluorography.

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