Figure 7.

Tim23p lacking the fourth TM segment is not efficiently imported into mitochondria. (A) Tim23p lacking the fourth TM segment is incompletely imported into mitochondria. Constructs of Tim23p lacking the fourth TM segment (Δ4) or lacking loop L3 and the fourth TM segment (ΔL3Δ4) were generated. Schematic representations of the constructs are shown to the left of the gel. The three proteins were synthesized and incubated with mitochondria. After the import reaction, samples were either analyzed directly (mitos) or after treatment with 200 μg/ml trypsin (mitos + trypsin) or 200 μg/ml proteinase K (mitos + proK). Mitochondrial pellets were analyzed by SDS-PAGE and fluorography. (B) The fourth TM segment and loop L3 are not required for Tim23p function. tim23::URA3 trp1 leu2 cyh2 strain KRR146 carrying TIM23-TRP1-CYH2 plasmid pKR1 was transformed with six different plasmids: LEU2-containing plasmid pJE50, which expresses wt Tim23p; pAD75, which expresses Tim23p lacking the fourth TM segment (TM4); pKR2, which lacks loop L3 and TM4; pKR34, which lacks the TM3 and TM4; pKR40, which lacks TM2, TM3, and TM4; and the empty vector pRS315. Leu+ colonies were patched out onto synthetic complete medium lacking leucine (SD−Leu). Patches were grown at 30°C for 2 d, and then replica-plated onto YEPD medium containing 10 mg/L cycloheximide (YEPD + CYH). Yeast cells that contain a functional Tim23 protein are able to lose the TIM23-TRP1-CYH2 plasmid and grow in the presence of cycloheximide.