Figure 1. SLC25A39 (A39) is a short-lived protein, and the degradation is regulated by GSH and the matrix cysteine residues.

(A) AlphaFold prediction of A39 structure revealed a unique cysteine-rich matrix loop 1.

(B) Western blot of endogenous A39 and ectopically expressed FLAG-tagged A39^WT, substrate binding pocket mutant A39^C334S, and matrix loop 1 four cysteine mutant A39^4CA in the K562 cells upon GSH depletion by BSO treatment (1mM, 2d). GAPDH, loading control.

(C) Mitochondrial GSH level assayed by HA-MITO tag immunoisolation followed by MS-based metabolite profiling in the control and clonal A39 CRISPR KO K562 cells expressing the indicated A39 constructs. 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.

(D) Scatter plot showing the correlation of protein abundance (log₁₀ nM) and mRNA abundance (Log₁₀ TPM) for all coding genes expressed in the HEK293 cells (OpenCell, CZI). Blue dots, SLC25 transporter proteins. A39 and A40 are highlighted in red.

(E-F) Western blot showing A39-FLAG (E) and A39^4CA-FLAG (F) protein level in the clonal A39 CRISPR KO K562 cells treated with CHX (cycloheximide, 150 μg/ml) for the indicated times, either under basal condition (−BSO) or with BSO (1mM and with 2 d prior treatment). GAPDH, loading control. A39 band intensity was quantified and modeled using the non-linear fitting with one phase decay (A39, −BSO; A39^4CA, −BSO and +BSO) and plateau followed by one phase decay (A39, +BSO).

See also Figure S1.