(a) Confirmation of PSD1 overexpression by qPCR analysis for the samples used in Fig. 2f. n = 3 biological replicates; One-way ANOVA followed by Dunnett’s test. (b) Cells expressing P TEF2 -V5-PDR3 and control no-V5 cells (P TEF2 -GFP-PDR3) were incubated for 4 h under control or protein import stress conditions (induced by PSD1 overexpression). V5-Pdr3 was immunoprecipitated using V5-Trap beads. Untagged Mge1 was detected using Mge1 antiserum. The asterisks indicate nonspecific bands. OE, overexpression. p, precursor form. m, mature form. The corresponding PSD1 mRNA levels are shown on the right panel. n = 3 biological replicates; one-way ANOVA followed by Dunnett’s test. (c) Cells expressing MGE1-FLAG and cells expressing MGE1-FLAG and P TEF2 -GFP-PDR1 were incubated for 4 h under control or protein import stress conditions. GFP-Pdr1 was immunoprecipitated using anti-GFP antibodies coupled to IgG beads from cells expressing P TEF2 -GFP-PDR1. The asterisk indicates a nonspecific band and ▲ indicates the GFP antibody’s light chain. OE, overexpression; p, precursor form; m, mature form. The corresponding PSD1 mRNA levels are shown on the right panel. n = 3 biological replicates; one-way ANOVA followed by Dunnett’s test. (d) Confirmation of PSD1 overexpression by qPCR analysis for the samples used in Fig. 2g. n = 3 biological replicates; one-way ANOVA followed by Tukey’s test. (e) Serial dilutions of wild-type and rho− cells. Cells were grown to logarithmic phase and spotted on YP (1% yeast extract, 2% bactopeptone) plates supplemented by 2% glucose or 3% glycerol. (f) CIS1 mRNA levels in wild-type and rho− cells in the presence of absence of PDR3. n = 3 biological replicates; one-way ANOVA followed by Dunnett’s test. (g) In vitro protein import assay using mitochondria isolated from wild-type and rho− cells. Mitochondria were incubated with radiolabeled 35S-Mdh1 and 35S-Mge1 for the indicated durations. Membrane potential (Δψ) was dissipated by preincubation with antimycin A, oligomycin, and valinomycin. Quantifications from three biological replicates are shown on the right; protein levels imported into wild-type mitochondria at the final time point were set to 100%. p, precursor; m, mature. (h) Immunoblot analysis of Mge1-FLAG and untagged Mge1 in wild-type and rho− cells. Wild-type cells that do not contain a FLAG tag were used as a control for Mge1-FLAG. p, precursor form. m, mature forms. (i) Half-life analysis of the Mge1-FLAG precursor in rho− and PDR3-deleted rho− cells. Cells were treated with cycloheximide (CHX, 0.5 mg/ml) and samples were collected at the indicated time points. Quantification of 4 biological replicates is shown on the right. p, precursor form. m, mature form. (j) Same as (i). Half-life analysis of the untagged Mge1 precursor in rho− and PDR3 deleted rho− cells (n = 3 biological replicates). p, precursor form. m, mature form. Asterisks indicate nonspecific bands. (k) Same as in (i), rho− cells were treated with DMSO (vehicle control) or MG132 (40 µM) for 1 h prior to the addition of cycloheximide (n = 3 biological replicates). (a-d, f, g, i-k) Data represent mean +/− SD. (a-d) **** P ≤ 0.0001.
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