Figure 3.

The preproteins of Tim22 and Tim54 use different Tim pathways for their import. Mitochondria were incubated in the presence or absence of b₂(167)Δ-DHFR and methotrexate for 15 min at 25°C. Mitochondria were reisolated, washed, and resuspended in import buffer containing methotrexate before the addition of ³⁵S-labeled (A) Tim22, (B) Tim54, (C) the ADP/ATP carrier (AAC), or (D) the Fe/S protein (Fe/Sp). Import proceeded for the indicated times, and mitochondria were subsequently treated with 50 μg/ml proteinase K. Where indicated, the membrane potential (Δψ) was dissipated before the addition of preprotein. Mitochondrial proteins were separated by SDS-PAGE and radiolabeled proteins were detected by storage phosphorimage cassette technology. Processing of the presequence of the Fe/S protein by matrix-located proteases generates intermediate (i) and mature (m) forms as indicated. The amount of imported preprotein was quantitated using ImageQuant software. The import in the absence of b₂(167)Δ-DHFR after the longest import time was set to 100% for each preprotein. (E) Tim22, Tim54, and AAC preproteins were imported into mitochondria for 30 min in the presence or absence of accumulated b₂(167)Δ-DHFR and subjected to blue native PAGE. Assembled preprotein was quantitated using ImageQuant software where 100% was set to preprotein assembled in the absence of b₂(167)Δ-DHFR.