Figure 1.

Identification of Tim50 crosslinking partners. (A) Schematic representation of Tim50 and Tim50 fragments used in this study. Presequence (PS), transmembrane domain (TM), NIF-domain, presequence-binding domain (PBD), and the cysteine position (C268) are indicated. (B) Structure of Tim50^C (Protein Data Bank entry 3QLE) with the Tim23-binding site, large negatively charged groove and the cysteine (C268, in green) positions indicated. Positively charged residues are coloured in blue, negative are red. (C) Wild-type and Tim50^HA-mitochondria were subjected to CuSO₄ crosslinking and analysed by western blotting using either anti-Tim50 or anti-HA antibodies. Selected shifted crosslinking products are marked with circles or a diamond (unknown Tim50 crosslinking partner). (D) Crosslinking was performed as in (C) using Tim21^ProtA-, Tim23^ProtA- and Tim23^ProtA-mitochondria. Samples were analysed by western blotting using anti-Tim50 antiserum. (E) CuSO₄ crosslinking was performed in mitochondria from Tim21^ProtA and tim21Δ strains, transformed with either an empty pFL39 (control) or a Tim21-expressing plasmid (TIM21). Samples were analysed by western blotting using an antiserum against Tim50.