Figure 3. A39 degradation is targeted through the matrix loop 1, which controls mitochondrial glutathione uptake and steady state level.

(A) Sequence alignment showing matrix loop 1 of A39, SLC25A40, and other representative SLC25 transporters SLC25A15, SLC25A1 and SLC25A11.

(B) Diagram showing Loop 1 swapping chimeric constructs.

(C) FLAG western blot for the indicated constructs in the clonal A39 CRISPR KO K562 cells, upon AFG3L2 CRISPR KO (+sgAFG3L2). GAPDH, loading control.

(D) Western blot of endogenous A39 in the whole cell lysates and mitochondrial lysates from the control and AFG3L2 CRISPR KO K562 cells. Mitochondrial protein VDAC, loading control.

(E) Mitochondrial GSH level assayed by HA-MITO tag immunoisolation followed by MS-based metabolite profiling in the control and AFG3L2 CRISPR KO K562 cells, normalized by total mitochondrial protein abundance.

(F) Western blot of A39 from BSO (1mM, 2 d)-treated clonal A39 CRISPR KO cells expressing either wild type A39 and A39^4CA-FLAG. VDAC, loading control.

(G) GSH uptake (1mM labeled GSH + 4mM unlabeled GSH, 15 min at room temperature) into HA-MITO immunoisolated mitochondria from BSO-treated clonal A39 CRISPR KO cells expressing either wild type A39 or A39^4CA-FLAG.

(H) Western blot of A39^WT and A39^A11L1-FLAG in the clonal A39 CRISPR KO cells.

(I) Mitochondrial GSH level assayed by HA-MITO tag immunoisolation followed by MS-based metabolite profiling in control, A39 CRISPR KO, and A39 CRISPR KO re-expressing A39^WT, or A39^A11L1-FLAG, normalized by NAD⁺ level.

(J) GSH uptake (5mM labeled GSH, 15 min at room temperature) into isolated mitochondria from A39 CRISPR KO cells expressing A39^WT and A39^A11L1-FLAG. All statistical significance was calculated using two-tailed t test. Significance level was indicated as *** p < 0.001, ** p < 0.01. Data are expressed as mean ± SD.