Figure 5.

The IMS-domain of Tim23 facilitates interaction between Tim21^IMS and Tim50. (A, B) Cu²⁺ crosslinking was performed in wild-type and (A) mgr2Δ or (B) Tim23 shut down mitochondria (Tim23↓), followed by immunoblotting against Tim50. (C) Indicated amounts of mitochondria from wild-type and Tim23↓ strains were analysed by SDS–PAGE and immunoblotting. (D) Mitoplasts generated from wild-type or Tim23↓ mitochondria were subjected to crosslinking with Cu²⁺ after incubation with the indicated concentrations of Tim21^IMS. (E) Mitochondria were treated like in (D), but Tim23^IMS was added where indicated prior to crosslinking. (F) Tim50–Tim21^IMS crosslink intensities from independent experiments (performed as in E) after treatment with 20 μM Tim23^IMS (n=9) or buffer (n=11) were quantified. Data are presented as mean±s.e.m. (G) Purified Tim21^IMS was immobilized on CNBr-activated Sepharose and incubated with 20 nM Tim50^IMS in the presence of the indicated Tim23^{IMS WT} or Tim23^{IMS YL70AA} concentrations. Bound Tim50 was eluted with 0.1 M glycin, pH 2.5 and analysed by SDS–PAGE followed by immunoblotting against Tim50. (H) Quantification of Tim50^IMS signal intensities, as in (G) (presented as mean±s.e.m., n≥3).