Figure 4: Translocase recruitment of motor subunits is affected in mutant mitochondria.
(a) Experimental scheme assaying the assembly of radiolabelled subunits to the resting TIM23 translocase. (b) [35S]Pam16, [35S]Pam18 or [35S]Tim44 were imported into isolated tim21Δ or mgr2Δ and the corresponding WT mitochondria. After Δψ dissipation, an aliquot of the sample was proteinase K treated (imported fraction) or solubilized with digitonin and subjected to α-Tim23 co-immunoprecipitation. Samples were analysed by SDS–PAGE, digital autoradiography and western blotting. The quantification shows the assembly efficiency of the radiolabelled subunits (% of imported, normalized to the unlabelled protein and Tim23) in mutants as % difference to the respective WT control (mean±s.e.m., n=3, *a=0.05, **a=0.01, one-tailed t-test for unequal variances). (c) During polypeptide translocation TIM23 complex-associated motor subunits are recruited to the import channel. Recharging of the translocase with co-chaperones is required to support continuous precursor translocation.
