Extended Data Fig. 1: Arrested presequence precursors are in proximity to Tim17.
From: Central role of Tim17 in mitochondrial presequence protein translocation
a–c, Import of 35S-labelled b2(110)∆19-DHFR# and b2(84)+7-DHFR with cysteine residues at the indicated positions (Cys pos.) in wildtype (WT) or Tim172xStrep (2xS) and HisSUMOstarTim23 (HS*) mitochondria in the presence of methotrexate (MTX) followed by chemical crosslinking (XL) with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS, a) or bismaleimidoethane (BMOE, b and c). Samples were analysed by SDS-PAGE and autoradiography. b2∆-Tim17, b2(110)∆19-DHFR#-Tim17 crosslinking product; b2∆-Tim172xStrep, b2(110)∆19-DHFR#-Tim172xStrep crosslinking product; b2-Tim17, b2(84)+7-DHFR-Tim17 crosslinking product; b2-Tim172xStrep, b2(84)+7-DHFR-Tim172xStrep crosslinking product; p, precursor; i, intermediate. d, Radiolabelled b2(110)∆19-DHFR# (lanes 1–7) or Cox5a(1–130)-sfGFP (lanes 8–14) were imported into Tim17 WT, tim17-4, tim17-5 or Tim172xStrep (2xS) and HisSumo*Tim23 (HS*) mitochondria following heat shock. Crosslinked products were either directly analysed (b2(110)∆19-DHFR#, lanes 1–7) or purified using a GFP nanobody (Cox5a-sfGFP, lanes 8–14) prior to analysis by SDS-PAGE and autoradiography. Cox5a-Tim17, Cox5a-sfGFP-Tim17 crosslinking product; Cox5a-Tim172xStrep, Cox5a-sfGFP-Tim172xStrep crosslinking product; m, mature.
