The MTS of ATFS-1 is less import competent. (a–c) Comparison of import competency between su9-DHFR and ATFS-1-DHFR. The N-terminus 73 amino acids of ATFS-1 was used as MTS. glp-1(e2141ts) animals were grown at 25°C on bacteria expressing mrps-5 dsRNA (20% diluted with bacteria containing the empty RNAi vector alone) from hatching until the first day of adulthood. Mitochondria were isolated on day 1 of adulthood and subjected to import assay with 30-min incubation time, followed by Western blot analysis. Import efficiency was quantified by measuring the mature imported protein as detected by the DHFR antibody. Quantification of imported DHFR is shown in b and c. (b) Import of ATFS1-DHFR was compared between mrps-5 RNAi and empty vector control with unpaired Student’s t test (two-tailed). The graph is presented as mean ± SD, n = 4. **, P < 0.01. (c) Import of DHFR with the MTS of su9 and ATFS-1 were compared with Student’s t test (two-tailed). The graph is presented as mean ± SD, n = 3. *, P < 0.05; **, P < 0.01. (d) Mitochondrial targeting capacity abolished by point mutations et15 or et18 in the MTS of ATFS-1. Mitochondria extraction was made from synchronized N2 wild-type worms at day 1 of adulthood and subjected to import assay with different substrates. Import with et15 or et18 was below the detectable level. Solid arrowheads, mature (imported) su9-DHFR with the su-9 MTS cleaved off; open arrowheads, mature (imported) ATFS1-DHFR with the MTS of ATFS-1 cleaved off. Source data are available for this figure: SourceData F5.
Figure 5.

The MTS of ATFS-1 is less import competent. (a–c) Comparison of import competency between su9-DHFR and ATFS-1-DHFR. The N-terminus 73 amino acids of ATFS-1 was used as MTS. glp-1(e2141ts) animals were grown at 25°C on bacteria expressing mrps-5 dsRNA (20% diluted with bacteria containing the empty RNAi vector alone) from hatching until the first day of adulthood. Mitochondria were isolated on day 1 of adulthood and subjected to import assay with 30-min incubation time, followed by Western blot analysis. Import efficiency was quantified by measuring the mature imported protein as detected by the DHFR antibody. Quantification of imported DHFR is shown in b and c. (b) Import of ATFS1-DHFR was compared between mrps-5 RNAi and empty vector control with unpaired Student’s t test (two-tailed). The graph is presented as mean ± SD, n = 4. **, P < 0.01. (c) Import of DHFR with the MTS of su9 and ATFS-1 were compared with Student’s t test (two-tailed). The graph is presented as mean ± SD, n = 3. *, P < 0.05; **, P < 0.01. (d) Mitochondrial targeting capacity abolished by point mutations et15 or et18 in the MTS of ATFS-1. Mitochondria extraction was made from synchronized N2 wild-type worms at day 1 of adulthood and subjected to import assay with different substrates. Import with et15 or et18 was below the detectable level. Solid arrowheads, mature (imported) su9-DHFR with the su-9 MTS cleaved off; open arrowheads, mature (imported) ATFS1-DHFR with the MTS of ATFS-1 cleaved off. Source data are available for this figure: SourceData F5.

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