Fig. 3: DELE1 maintains mitochondrial presequence protein import upon stress. | Nature Communications

Fig. 3: DELE1 maintains mitochondrial presequence protein import upon stress.

From: The integrated stress response suppresses PINK1-dependent mitophagy by preserving mitochondrial import efficiency

Fig. 3: DELE1 maintains mitochondrial presequence protein import upon stress.The alternative text for this image may have been generated using AI.

a Immunoblots of HSPD1 and ATF4. HEK293T cells with an NTC or DELE1 sgRNA (DELE1 KD) were treated with 10 μM CCCP or 1.25 ng/mL oligomycin (Oli) for 24 h. p: precursor form of HSPD1. m: mature form of HSPD1. β-actin was used as a loading control. b Immunoblots of OTC-V5 (via anti-V5 antibody) and ATF4. HEK293T inducible OTC-V5 cells with an NTC or DELE1 sgRNA were treated 500 ng/mL doxycycline (Dox) concurrently with 10 μM CCCP or 1.25 ng/mL oligomycin (Oli) for 24 h. p: precursor form of OTC-V5. m: mature form of OTC-V5. β-actin was used as a loading control. Listed numbers in the bottom are the fractions of non-imported OTC-V5, calculated by dividing the level of precursor by the total (precursor + mature) protein level. c Left: Inducible MTSCOX8-YFP cells with an NTC or DELE1 sgRNA were treated with 500 ng/mL doxycycline (Dox) concurrently with 10 μM CCCP or 1.25 ng/mL oligomycin for 24 h. Arrowheads highlight cells with obvious diffused YFP signals. Scale bar 100 μm. Right: Quantification of percentage of cells with obvious diffused YFP signals following oligomycin treatment (mean ± s.d., n = 3, each with about 100 cells analyzed) d Schematic illustration for monitoring mitochondrial protein import using split-GFP (inducible MTS-GFP11+ constitutive GFP1-10) reporter via flow cytometry. e Measurement of the import of MTSCOX8-GFP11 in GFP1-10 cells with an NTC or DELE1 sgRNA following 1.25 ng/mL oligomycin or 10 μM CCCP treatments. Expression of MTSCOX8-GFP11 was induced simultaneously during mitochondrial toxins treatment by adding doxycycline. (mean ± s.d., n = 3 independently treated culture wells) Two-way ANOVA test followed by Turkey’s multiple comparisons test (two-sided). **** adjusted p value < 0.0001. f Measurement of the import of MTSDiablo-GFP11 in HEK293T cells with an NTC or DELE1 sgRNA. (mean ± s.d., n = 3 independently treated culture wells) Two-way ANOVA test followed by Turkey’s multiple comparisons test (two-sided). **** adjusted p value < 0.001. g Schematic illustration for monitoring mitochondrial protein import using split-GFP (inducible MTS-GFP11+ constitutive MTS-GFP1-10-degron) reporter via flow cytometry. h Measurement of the import of MTSCOX8-GFP11 in MTS-GFP1-10-degron cells with an NTC or DELE1 sgRNA following 1.25 ng/mL oligomycin or 10 μM CCCP treatments. Expression of MTSCOX8-GFP11 was induced simultaneously during mitochondrial toxins treatment by adding doxycycline. (mean ± s.d., n = 3 independently treated culture wells) Two-way ANOVA test followed by Turkey’s multiple comparisons test (two-sided). ** adjusted p value = 0.0012. i Cell free mitochondrial protein import assay using mitochondrial isolated from WT and DELE1 KD cells treated with oligomycin for 24 h. The mitochondria were incubated with MTS-DHFR-flag for the indicated time. To dissipate membrane potential, mitochondria were pre-incubated with CCCP for 5 min. Proteinase K (PK) was used to digest unimported substate post import reaction. Source data are provided as a Source Data file.

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