Figure 1: Preserving a TOM/TIM23-spanning translocation intermediate requires import motor activity. | Nature Communications

Figure 1: Preserving a TOM/TIM23-spanning translocation intermediate requires import motor activity.

From: Remodelling of the active presequence translocase drives motor-dependent mitochondrial protein translocation

Figure 1

(a) Model of TIM23 complex with associated import motor and a substrate (blue). (b) Isolated mitochondria were subjected to heat shock before import of purified b2(167)Δ-DHFR in the presence or absence of methotrexate (MTX). Samples were analysed by BN-PAGE and α-Tim23 western blotting. (c) Experimental scheme to assess maintenance of TOM/TIM23-spanning translocation intermediate in temperature-sensitive mutants. (d) Quantification of TOM/TIM23 translocation intermediates after motor inactivation. Amount of supercomplex retained after 37 °C chase (100%; supercomplex retained during 25 °C chase). (e) Import of [35S]Atp2 into isolated WT and mutant mitochondria (mean±s.e.m., n=3). (f) Import of sorted precursors into isolated WT and mgr2Δ mitochondria (mean±s.e.m., n=3). (g) Inward driving activity on arrested [35S]b2(220)-DHFR. After import in the presence of MTX, Δψ was dissipated before a second incubation at 25 °C for the indicated time. Samples were PK treated and the amount of the intermediate quantified (100%; processed intermediate without protease treatment, mean±s.e.m., n=3). (h) Inward driving activity assayed at 5 min chase time in indicated mutant and respective WT mitochondria as described in g (mean±s.e.m., n≥3).

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