Figure 1.

Membrane integration and assembly of Tim23p mutants. (A) Linear representations of the essential membrane proteins in the TIM23 complex. Large boxes denote proposed Tim23p TMS regions (TMS1-4); thin boxes denote the hydrophilic N-terminal region and loops (L1-3) in the IMS (shifted upward) and in the matrix (shifted downward). Residue numbers of Tim23p are shown at the top. Open ovals show native Cys positions in S. cerevisiae Tim23p (Cys98, Cys209, and Cys213) that were changed to Ala in the Tim23pΔCys construct; closed ovals show native Cys positions in Tim17p and Tim50p. Gray rectangles in Tim23p show positions of individual monocysteine mutations used in this study. Slash bars in the Tim50p cartoon are breaks between positions 100 and 140 and between positions 240 and 450. (B) Tim23p monocysteine mutants are imported into mitochondria and integrated into the IM with efficiency similar to wild-type. Wild-type, Cys-less (ΔCys), and the indicated monocysteine mutants of in vitro–translated [³⁵S]Tim23p were incubated with energized mitochondria or with mitochondria in which the membrane potential had been dissipated (−Δψ, lanes 5, 10, 15, 20, and 25) with 1 μM valinomycin. Samples were then subjected to the treatments after import: addition of protease directly to intact mitochondria (lanes 2, 7, 12, 17, and 22 for energized mitochondria, and lanes 5, 10, 15, 20, and 25 for −Δψ mitochondria); hypotonic swelling to produce mitoplasts and then addition of protease to produce the ∼14-kDa fragment (marked by the arrowhead) characteristic of membrane integration (“swelling”, lanes 3, 8, 13, 18, and 23); addition of the detergent Triton X-100 followed by addition of protease (“TX-100”, lanes 4, 9, 14, 19, and 24). Lanes 1, 6, 11, 16, and 21 show 20% of the total in vitro translation product added to each import reaction. (C) Tim23p monocysteine mutants are assembled into TIM23 complexes with efficiency similar to wild type. Wild-type (WT), G186D (corresponding to the Tim23-1 mutant; Lohret et al., 1997), ΔCys, and the indicated monocysteine mutants of [³⁵S]Tim23p were incubated with energized mitochondria or mitochondria with a dissipated membrane potential (WT −Δψ), then subjected to wash in a buffer containing 1% digitonin to maintain the TIM23 complex, and coimmunoprecipitated with antibodies directed against Tim17p (CoIP, lanes 2, 4, 6, 8, 10, 12, and 14). Lanes 1, 3, 5, 7, 9, 11, and 13 show 5% of total in vitro translation product added to each import reaction before membrane solubilization.