C. elegans mitochondrial protein in vitro import assay. (a) Schematic diagram of the C. elegans in vitro mitochondrial protein import assay. (b and c) su9-DHFR was transcribed and translated in a single reaction with the Quick Coupled Transcription/Translation System (TnT reaction). Mitochondria extraction was made from synchronized N2 wild-type worms at day 1 of adulthood and quantified with BCA analysis. 50 µg mitochondrial protein was used in each reaction. The substrate protein was incubated with mitochondria extraction in import buffer containing an ATP regeneration system for 10, 20, or 30 min at 25°C. Mitochondria were subsequently treated with proteinase K to remove non-imported proteins. Upon being imported, the MTS of su9 is cleaved. 2 µg/ml valinomycin was used to disrupt the membrane potential (ΔΨ), thus inhibiting import. The precursor (p) and mature protein (m) were detected with the DHFR antibody by Western blot analysis. Right lane: 20% of the su9-DHFR substrate used in the import assay representing the precursor (p). (d and e) Germline-deficient, mutant glp-1(e2141ts) worms were bleach synchronized, grown at the restrictive temperature of 25°C, and treated with RNAi against tomm-20 or timm-17 until the first day of adulthood. Control worms were grown on bacteria containing empty vector alone. Mitochondria were isolated and subjected to the import assay. 2 µg/ml valinomycin was used to disrupt the membrane potential (ΔΨ), thus inhibiting import. (c and e) The efficiency of mitochondrial import was quantified by measuring the mature imported protein as detected by the DHFR antibody and analyzed with unpaired Student’s t test (two-tailed). All graphs are presented as mean ± SD. *, P < 0.05. n = 3 (c); n = 2 (e). Arrowheads, mature (imported) DHFR with the MTS cleaved off. Source data are available for this figure: SourceData F1.
Figure 1.

C. elegans mitochondrial protein in vitro import assay. (a) Schematic diagram of the C. elegans in vitro mitochondrial protein import assay. (b and c) su9-DHFR was transcribed and translated in a single reaction with the Quick Coupled Transcription/Translation System (TnT reaction). Mitochondria extraction was made from synchronized N2 wild-type worms at day 1 of adulthood and quantified with BCA analysis. 50 µg mitochondrial protein was used in each reaction. The substrate protein was incubated with mitochondria extraction in import buffer containing an ATP regeneration system for 10, 20, or 30 min at 25°C. Mitochondria were subsequently treated with proteinase K to remove non-imported proteins. Upon being imported, the MTS of su9 is cleaved. 2 µg/ml valinomycin was used to disrupt the membrane potential (ΔΨ), thus inhibiting import. The precursor (p) and mature protein (m) were detected with the DHFR antibody by Western blot analysis. Right lane: 20% of the su9-DHFR substrate used in the import assay representing the precursor (p). (d and e) Germline-deficient, mutant glp-1(e2141ts) worms were bleach synchronized, grown at the restrictive temperature of 25°C, and treated with RNAi against tomm-20 or timm-17 until the first day of adulthood. Control worms were grown on bacteria containing empty vector alone. Mitochondria were isolated and subjected to the import assay. 2 µg/ml valinomycin was used to disrupt the membrane potential (ΔΨ), thus inhibiting import. (c and e) The efficiency of mitochondrial import was quantified by measuring the mature imported protein as detected by the DHFR antibody and analyzed with unpaired Student’s t test (two-tailed). All graphs are presented as mean ± SD. *, P < 0.05. n = 3 (c); n = 2 (e). Arrowheads, mature (imported) DHFR with the MTS cleaved off. Source data are available for this figure: SourceData F1.

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