Figure 3.

Interaction of presequence peptides with Tim50. (A–F) Purified 6 × His-tagged Tim50^IMS (A, B), Tim50^PBD (C, D), or Tim50^C (E, F) was immobilized on a Ni²⁺-chelator chip and the SPR response to the indicated concentrations of pALDH (A, C, E) or pALDH-s (B, D, F) was recorded. K_D is presented as mean±s.e.m. (n=3 for each measurement). (G) Photocrosslinking was performed using purified 1 μM Tim50^IMS, Tim50^ΔPBD, Tim50^C and biotinylated photopeptides pL₁₉B and pS₁₆B at indicated concentrations. After SDS–PAGE separation, crosslinking products were detected using streptavidin–horse radish peroxidase (SA-HRP) conjugate or by immunodecoration against Tim50. (H) HA-tagged full-length Tim50 (Tim50^HA) and Tim50 fragments containing the first 365 or 361 amino acids (Tim50^1–365–HA, Tim50^1–361, and Tim50^1–361–HA) in pME2782 were used to replace the wild-type protein-encoding plasmid carrying URA3 in the gene deletion strain grown on 5-fluoroorotic acid-containing plates (SD-5FOA). Strains were grown on selective-Leu plates as a control.