Figure 6.

Efficient import of Tim23p requires a pair of hydrophobic segments. (A) Further deletions of the Δ1Δ2 protein inhibit import. The Δ1Δ2 construct contains the third and fourth TM segments of Tim23p, as well as the intervening loop L3. Constructs lacking the fourth TM segment, lacking loop L3 and the fourth TM segment, or lacking loop L3 and the third TM segment were generated as described in MATERIALS AND METHODS. Schematic representations of these constructs are shown on the left side of the gel. The four proteins were synthesized and imported into isolated mitochondria either in the presence or absence (−ΔΨ) of a membrane potential. After import, samples were either not treated (mitos) or treated with 200 μg/ml trypsin (mitos + trypsin; −ΔΨ). Mitochondrial pellets were analyzed by SDS-PAGE and fluorography. (B) Further deletions of the Δ3Δ4 protein inhibit import. The Δ3Δ4 construct contains the first and second TM segments of Tim23p, as well as the intervening loop L1. A construct lacking the second TM segment and loop L1, or a construct lacking the first TM segment and loop L1, was produced. Diagrams illustrating the constructs are shown to the left of the gel. The three proteins were synthesized and imported either in the presence or absence (−ΔΨ) of a mitochondrial membrane potential. After import, samples were either not treated (mitos) or treated with 200 μg/ml trypsin (mitos + trypsin; −ΔΨ) before analysis.