Role of GCN1L1-GCN2-ATF4 pathway in mammalian mitochondrial retrograde signaling Ken Itoh, Shuya Kasai 1Department of Stress Response Science, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
Mitochondria is an organelle that undertakes an efficient energy production in the cells by oxidative phosphorylation and its disturbances are involved in many age-related diseases due to the lowered ATP production or the overproduction of reactive oxygen species. Retrograde signaling acts primarily to improve the mitochondrial perturbation and considered as a homeostatic stress response against intrinsic and extrinsic stimuli. There are several branches of the mitochondrial retrograde signaling including mitochondrial unfolded protein response, but recent observations increasingly show the importance of ISR-ATF4 pathway in the mitochondrial retrograde signaling. In this study, we examined the role of GCN1L1-GCN2 pathway using human haploid cell line HAP-1 that lack GCN1L1 (GCN1L1-deficient HAP-1 cells). We used the induction of asparagine synthase (ASNS) gene as a maker of ATF4 activation. ASNS gene induction by histidinol that mimic histidine starvation in cells was observed in wild-type HAP-1 cells, but not in GCN1L1-deficient HAP-1 cells, confirming that GCN1L1 is essential in the amino acid response as reported about yeast GCN1. Mitochondrial disturbance by oligomycin and doxycycline efficiently induced ASNS gene expression in wild-type HAP1 cells. However, ASNS gene induction by doxycycline was decreased about by half in GCN1L1-deficient HAP-1 cells compared to that in wild-type HAP1 cells, although the induction by oligomycin was comparable between the cells. Phosphorylation of eIF2ɑ by histidinol and doxycycline was decreased in GCN1L1-deficient HAP-1 cells compared to that in wild-type HAP1 cells. GCN2 phosphorylation was only apparent in cells treated by histidinol. These results indicated that GCN1L1 is selectively involved in the ASNS gene induction by mitochondrial stress induced by doxycycline, but not by oligomycin. We also examined the ATF4 activation status cultured in high glucose medium or glucose-free medium with galactose and high glutamine (OxPhos medium). Shifting the cells to OxPhos medium induced ASNS gene induction only in GCN1L1-deficient HAP-1 cells, indicating that GCN1L1 somehow ameliorates mitochondria-derived stress in OxPhos medium. Thus, GCN1L1 is involved in mitochondrial stress response in a multiple and specific manners.
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