Fig. 3
From: The selectivity filter of the mitochondrial protein import machinery
Binding of [35S]-labeled polypeptides to the purified cytosolic domain of yeast Tom70 and to isolated mitochondria. a SDS-PAGE of 0.75 μg isolated Tom70cd (cytosolic domain, residues 38–617). b Binding of [35S]-labeled polypeptides to Tom70cd. Purified Tom70cd was pre-bound to Ni-NTA beads and incubated with reticulocyte lysate containing radiolabeled wild type AAC (AACWT), or different segments of the AAC fused to DHFR: C-terminal part of AAC module III including the matrix loop and helix 6 (ML3-TMD6, residues 238–313), the matrix loop (ML3, residues 238–277), helix 6 (TMD6, residues 277–313), or the AAC derivative lacking the positively charged residues in the matrix loops (AACΔpos). Bound radiolabeled proteins were quantified using the phosphorimager, SD, n = 3. c Binding of [35S]-labeled AACWT to isolated Tom70cd pretreated with N-ethylmaleimide (NEM). The assays were performed as in b, but the Ni-NTA-bound Tom70cd was preincubated with up to 4 mM NEM as indicated. The amount of bound protein in absence of NEM was set to 100%. SD, n = 4. d Binding of [35S]-labeled AACWT to mutant versions of Tom70cd. Single cysteines in positions 439, 450, or 141, respectively, were exchanged against alanine. The modified Tom70cd proteins were bound to Ni2+-NTA, and as indicated, some samples were treated with 2 mM NEM. All beads were washed with assay buffer and subsequently used for binding assays as in c. SD, n ≥ 6. e Location of cysteine residue C141, arginine R171, and serine S174 in Tom70. The structural data were taken from Wu and Sha [53], PDB 2GW1. f Binding of [35S]-labeled yeast AAC2 to isolated mitochondria containing wild type Tom70. Radiolabeled AAC2 was synthesized in reticulocyte lysate and preincubated with apyrase; the mitochondria were pretreated with 2 mM NEM (+ NEM) or left untreated (− NEM). Binding in absence of NEM within 12 min was set to 100%, SD, n = 3. g Binding of [35S]-labeled AAC2 to mitochondria isolated from a yeast strain expressing Tom70C141A. The assay was carried out as in f. Binding in absence of NEM within 12 min was set to 100%, SD, n = 3. h Binding of AAC to mitochondria pretreated with NEM and trypsin, SD, n = 3