Loss of the mitochondrial protein Abcb10 results in altered arginine metabolism in MEL and K562 cells and nutrient stress signaling through ATF4

Abcb10 is a mitochondrial membrane protein involved in hemoglobinization of red cells. Abcb10 topology and ATPase domain localization suggest it exports a substrate, likely biliverdin (Shum et al Sci Transl Med 13, (2021), PMC8300486), out of mitochondria that is necessary for hemoglobinization. In this study we generated Abcb10 deletion cell lines in both mouse (Murine Erythroleukemia (MEL) and human erythroid precursor (Human Myelogenous Leukemia K562) cells to better understand the consequences of Abcb10 loss. Loss of Abcb10 resulted in an inability to hemoglobinize upon differentiation in both K562 and MEL cells with reduced heme and intermediate porphyrins and decreased levels of Aminolevulinic acid synthase 2 activity. Metabolomic and transcriptional analysis revealed that Abcb10 loss gave rise to decreased cellular arginine levels, increased transcripts for cationic and neutral amino acid transporters with reduced levels of the citrulline to arginine converting enzymes Argininosuccinate synthetase and Argininosuccinate lyase. The reduced arginine levels in Abcb10 null cells gave rise to decreased proliferative capacity and excess arginine supplementation improved both Abcb10 null proliferation and hemoglobinization upon differentiation. Abcb10 null cells showed increased phosphorylation of Eukaryotic Translation Initiation Factor 2 Subunit Alpha, increased expression of nutrient sensing transcription factor ATF4 and downstream targets DNA damage inducible transcript 3 (Chop) and ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (Chac1) and Arginyl-tRNA Synthetase 1 (Rars). These results suggest that when the Abcb10 substrate is trapped in the mitochondria, the nutrient sensing machinery is turned on remodeling transcription to block protein synthesis necessary for proliferation and hemoglobin biosynthesis in erythroid models. RNAseq expression profiling of WT MEL cells and Abcb10 knockout MEL cells differentiated for 0, 2 and 5 days in 1.5% DMSO.