Fig 1: The MTS facilitates Hsp90 retention on mitochondrial precursors.(A) pOTC-TST was translated in RRL in the presence or absence of SP cells and valinomycin (1 μM) and/or geldanamycin (2 μM). Total (top) or affinity purified (bottom) samples were analyzed by SDS–PAGE and autoradiography. pOTC is marked with blue arrows, whereas mature OTC with cleaved MTS is marked with red arrows. Note that proteasome-mediated degradation is not active in the RRL system, which is why geldanamycin treatment does not lead to destabilization of the precursor as seen in cells. (B) Full-length (FL) or ΔMTS OTC was translated in RRL in the presence or absence of 2 μM geldanamycin and affinity purified under native conditions via TST. Eluted samples were analyzed by immunoblotting alongside input samples. (C) Strategy to identify MTS-binding factors. Photoactivatable cross-linking amino-acid BpA was introduced into different positions within the MTS of pOTC using amber suppression. Amber mutants were translated in RRL and cross-linking was induced with UV. Cross-linking products were analyzed by SDS–PAGE. Two major cross-linking bands representing a 15–25 kDa adduct (light blue arrows) and a 40–60 kDa adduct (dark blue arrows) are marked. (D) pOTC with BpA at positions F3 or H18 was produced in RRL and cross-linked with UV. Samples were then immunoprecipitated with antibodies against Hsc70 or Hsp90α under native conditions and analyzed by SDS–PAGE. Major cross-linking bands are marked with the same colors as in C. Coomassie stained gel (bottom) verifies efficient pulldown of Hsc70 (orange arrows) and Hsp90 (red arrows). (E) BpA-containing pOTC mutants were produced in RRL and cross-linked. Fully denatured samples were then immunoprecipitated with antibodies against p23 and an unrelated protein of similar size (Btf3), and analyzed by SDS–PAGE. (F) Cells with stably integrated pOTC-GFP-P2A-RFP reporter were treated with control siRNA or siRNA targeting p23 for 3 d, and expression of the reporter was induced for another 7 h in the presence of indicated drugs. Cells were then analyzed by flow cytometry (top) or immunoblotting (bottom). Histogram representing GFP:RFP ratio is shown. See also Supplemental Figure S2.