Fig. 4: CHAC1 loss diminishes Fe-S protein function under Cys2 starvation. | Nature Communications

Fig. 4: CHAC1 loss diminishes Fe-S protein function under Cys2 starvation.

From: Mitochondrial respiratory function is preserved under cysteine starvation via glutathione catabolism in NSCLC

Fig. 4

a Schematic of the intracellular GSH cleavage system; Cys, cysteine, Cys2, cystine, Cys-Gly, cysteinylglycine, GGC, γ-glutamylcysteine, Gly, glycine, Glu, glutamate, GSH, glutathione. b Immunoblot analysis of CHAC1, CHAC2, ATF4, and α-Tubulin expression in NSCLC cells under cystine starvation. c Immunoblot analysis of CHAC1, CHAC2, ACO1, CS, MCU, TOM20, Grp94, Erp44, Catalase, and Lamin B2 expression in whole cell or fractionated lysates upon mitochondrial immunoprecipitation from cells cultured in the indicated [cystine] for 20 h. d Immunoblot analysis of TOM20, AIF, MICU1, NNT, NDUFS1, ACO2, CS, and CHAC1 in mitochondrial isolates subject to proteinase K treatment in the presence or absence of detergent; IMM inner mitochondrial membrane, IMS intermembrane space, OMM outer mitochondrial membrane. e ACO2 (n = 3 biologically independent samples per condition, except when n = 2 for sgControl-96h) and (f), supercomplex I–III activities (n = 3 biologically independent samples per condition) in CHAC1 expressing or deficient H1299 cells following treatment with cystine-deficient media supplemented with 1 μM ferrostatin-1 for the indicated time. g Immunoblot analysis of ACO2, NDUFS1, FECH, LIAS, SDHB, CHAC1, and β-Actin in CHAC1 expressing or deficient H1299 cells following culture in cystine-deficient media supplemented with 1 μM ferrostatin-1 for the indicated time. h Immunoblot analysis of CHAC1 and β-Actin expression in control or CHAC1-deficient H1299 cells reconstituted with vector control, untargeted, or mitochondrially-targeted CHAC1. i Immunoblot analysis of CHAC1, ACO1 (cytosol), and CS (mitochondria) expression in fractionated lysates from cells in (h); C cytosol, M mitochondria, WC whole cell. j ACO2 activities (n = 3 biologically independent samples per condition) and (k, l) ETC supercomplex activities (n = 6 biologically independent samples per condition) in control or CHAC1-deficient H1299 cells reconstituted with vector control, untargeted, or mitochondrially-targeted CHAC1 following 20 h culture with or without cystine. For (e, f) data points are representative of the activity of each Fe-S protein relative to cystine-replete conditions (time 0 h). Data represent mean values ± s.d. For (j–l) data are representative of at least 3 experimental replicates and P values were calculated using a two-way ANOVA. a created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license, https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. For (b–l), source data is provided as a Source Data file.

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