Figure 2. Co-immunoprecipitation mass spectrometry and CRISPR KO identify the mitochondrial m-AAA protease AFG3L2 in A39 degradation.

(A) Left, flow chart showing the experimental procedure to identify putative A39-interacting regulatory proteins. Right, silver stained SDS-PAGE of anti-FLAG immunoprecipitants from the control and A39-FLAG mitochondrial lysates. Wild type K562 cells expressing 3HA-eGFP-OMP25 (HA-MITO tag) were used as control. A39-FLAG protein identified by MS is labeled; *, a specific binding partner HSPD1 identified by MS.

(B) Categorization of specific A39-interacting protein hits into relevant quality control pathways. Protein level intensity based on top three peptides was shown in the control and A39-FLAG co-immunoprecipitants. Bold, hits that were followed up by CRISPR KO.

(C) Western blot of A39 from CRISPR KO K562 cells using sgRNAs targeting indicated A39-interacting proteins, and related mitochondrial proteases. Cells were analyzed under basal condition and BSO (200 μM) treatment for either 1 d or 2 d. Two CRISPR KO lines that increased basal A39 level were highlighted in red. GAPDH, loading control.

(D-E) Western blot of endogenous A39, ectopically expressed FLAG-tagged A39^WT and A39^4CA in the AFG3L2 CRISPR KO cells (+ sgAFG3L2, D) and ABCB7 CRISPR KO cells (+sgABCB7, E).

(F) Western blot of endogenous A39 in HeLa cells upon AFG3L2 CRISPR KO and re-expressing AFG3L2–3xFLAG. GAPDH, loading control.

(G) Western blot of the endogenous A39 protein in the K562 cells treated with CHX (cycloheximide, 150 μg/ml) for the indicated times, with or without AFG3L2 CRISPR KO. GAPDH, loading control.

(H) A39 band intensity was quantified and modeled by the non-linear fitting with one phase decay.

See also Figure S2 and Table S1.