Extended Data Fig. 7: The N-terminus of Mge1 contains critical features for the activation of the mitoCPR.

(a) Amino acid alignment and sequence conservation of Mge1 across 10 Saccharomyces species. (b) Immunoblot of overexpressed full-length and truncated Mge1-mCherry variants (4 h galactose induction). OE, overexpression. (c) In vitro protein import assay using mitochondria isolated from control cells (empty vector) and cells overexpressing MTS^Mge1(1-20)-mCherry for 4 h. Mitochondria were incubated with radiolabeled ³⁵S-Zim17 for the indicated durations. Membrane potential (Δψ) was dissipated by preincubation with antimycin A, oligomycin, and valinomycin. Quantification from three biological replicates is shown in the bottom panel; protein levels imported into control mitochondria at the final time point were set to 100%. p, precursor; m, mature. OE, overexpression. (d) Growth curves of cells overexpressing MTS¹¹⁶-mCherry or MTS^Mge1(1-20)-mCherry, in the presence or absence of PDR3, were plotted on a semilogarithmic scale. Corresponding growth rate (r) was calculated from the growth curves of three biological replicates, each with three technical replicates; two-tailed Student’s t-test. OE, overexpression. (e) Immunoblot of overexpressed MTS¹¹⁶-mCherry and MTS^Mge1(1-20)-mCherry 4 h following galactose addition. OE, overexpression. (f) Upper panel- Immunoblot of the endogenously expressed addbacks (full-length Mge1-mCherry, MTS^Mge1-mCherry, and MTS^Mge1(1-20)-mCherry) into the MTS^SU9-MGE1-FLAG background. Lower panel- Confirmation of PSD1 overexpression by qPCR analysis for the samples used in the upper panel and in Fig. 5c. n = 3 biological replicates; one-way ANOVA followed by Tukey’s test. OE, overexpression. (g) The electrostatic surface potential plot shows that the presequence of Mge1(residues 1–60, in green) fits into a negatively charged patch in Pdr3 (residues 86–856). (h-i) Heatmap of the amino acid enrichment across the first twenty amino acid of the MTSs from all yeast mitochondrial proteins (h) or previously reported nuclear-localized mitochondrial proteins (i). The enrichment score represents the frequency of a residue at a given position across the set weighted by the genome-wide amino acid frequencies. A table listing the N-terminal twenty amino acids of the mitochondrial presequences used in (i) is shown on the right. (j) Immunoblot of FLAG-tagged wild-type and mutants Mge1, expressed from the native MGE1 promoter, in the MTS^SU9-MGE1-FLAG background. The asterisk indicates a likely degradation product. p1, MTS^Su9-Mge1-FLAG precursor; p2, Mge1-FLAG variants precursor; m1/2, mature form MTS^Su9-Mge1-FLAG/Mge1-FLAG variants. OE, overexpression. (k) Confirmation of PSD1 overexpression by qPCR analysis for the samples used in Fig. 5f. n = 3 biological replicates; one-way ANOVA followed by Tukey’s test. OE, overexpression. (c, d, f, k) Data represent mean +/− SD; **** P ≤ 0.0001.

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