FIGURE 7.

MB-10 inhibits Tim44 binding to the precursor and to Hsp70, but not other components of the PAM complex. A, radiolabeled cyt b₂ (167)A63P-DHFR was imported into mitochondria isolated from WT or the tim44-8 mutant in the presence of 1 μm MTX. Reactions were treated with cross-linker 200 μm DSS. An asterisk indicates the Tim44-cyt b₂(167)A63P-DHFR cross-link that is absent in mitochondria from the tim44-8 mutant strain (22). p, precursor; i, intermediate. B, 20 μg of mitochondrial lysate was treated with 1% DMSO or 100 μm MB-10 and immunoprecipitated with anti-mitochondrial Hsp70 antibody in the absence of or presence of 5 mm MgCl₂ and 1 mm ATP (+ATP/Mg). Tim44 was detected with anti-Tim44 antibody, and mtHsp70 was visualized by Ponceau S staining. An asterisk indicates the IgG heavy chain. C, mitochondria (400 μg) from a strain in which Tim44 contained a C-terminal His₁₀ tag and a WT control were treated with 1% DMSO or 100 μm MB-10, followed by lysis in 1% digitonin. Tim44-His₁₀ was purified with Ni²⁺ agarose, and immunoblot analysis was performed with antibodies against Tim44, Pam16, Pam18, and Tom70. 50 μg of input (T) and flow through (FT) and 300 μg of eluate (E) were loaded. D, schematic showing the TIM23 translocon and associated PAM complex in the absence of MB-10 (left panel). MB-10 addition blocks interactions between Tim44 and the precursor and increases interactions between Hsp70 and the precursor while blocking translocation into the matrix (right panel, marked with an X). STD, standard.