Extended Data Fig. 8. Interaction of precursor proteins with the lateral Tim17 cavity is membrane potential-dependent.

a, Growth phenotypes of the indicated tim17 (top panel) or tim23 (bottom panel) cysteine mutants in the tim17∆tim23∆ background on YPG agar at 23 °C. b-e, Arrest of ³⁵S-labelled Cox5a(1–130)-sfGFP (b and e), b₂(84)₊₇-DHFR (c) or b₂(110)_∆19-DHFR^# (d) into Tim17^SCF and Tim23^CF mitochondria with cysteine residues introduced at the indicated positions. Chemical crosslinking (XL) was performed with MBS followed by SDS-PAGE and autoradiography. f, Methotrexate (MTX) preincubated, radiolabelled b₂(84)₊₇-DHFR or b₂(110)_∆19-DHFR^# was imported into Tim17^SCF or Tim17^SCF_N16C mitochondria in the presence or absence of a membrane potential (∆ψ) across the inner membrane followed by chemical crosslinking (XL) with MBS. Samples were subsequently analysed by SDS-PAGE and autoradiography. g,h, Radiolabelled b₂(84)₊₇-DHFR (g) or b₂(110)_∆19-DHFR^# (h) constructs with cysteine residues at the indicated positions were imported into Tim17^SCF+Tim23^CF control or mutant mitochondria with cysteine residues at the indicated position (Cys. pos.) within the transmembrane cavity. Import was followed by chemical crosslinking (XL) with BMOE, SDS-PAGE and autoradiography.