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. 2013 Feb 12;32(6):886–898. doi: 10.1038/emboj.2013.23

Figure 4.

Figure 4

Presequence peptides affect interactions between Tim21, Tim23, and Tim50. (A) Mitochondria were pre-treated with indicated concentrations of pALDH or its inactive version pALDH-s (reaction volumes were matched by adding corresponding volumes of solvent), crosslinked by 1 mM CuSO4 for 30 min on ice and analysed by non-reducing SDS–PAGE and immunodecoration against Tim50. (B) Signals of Tim23–Tim50 (upper plot) and Tim21–Tim50 (lower plot) crosslinking-adducts (as in A) were quantified, normalized to buffer-treated controls (set as 100%), and presented as mean±s.e.m. (n=6). (C) Experiment (as in A) was performed in osmotically swollen mitochondria (mitoplasts, n=4), mitochondria with a dissipated membrane potential (–Δψ, n=4), tom22-2 (n=4) and Tim501–361–HA (n=3) mitochondria. The intensity of the Tim21–Tim50 crosslink-adducts after treatment with 50 μM pALDH was quantified and normalized to control (set to 100%). Data are presented as mean±s.e.m. (D) Purified Tim21IMS and Tim50IMS (1 μM each) were crosslinked in the presence of increasing amounts of pALDH or pALDH-s and analysed by immunoblotting against Tim21.