Yeast strains used in this study.

A key mechanism for regulating the initiation of protein synthesis in response to various stresses involves the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Schizosaccharomyces pombe possesses three distinct eIF2α kinases: Hri1, Hri2, and Gcn2. Using a strain that is unable to synthesize heme de novo (hem1Δ), global transcriptome analysis reveals that among the genes encoding these kinases, hri1+ is the most strongly induced under δ-aminolevulinate (ALA)-limiting conditions. The induction of hri1+ consistently correlates with increased eIF2α phosphorylation and a reduction in global protein translation in ALA-starved hem1Δ cells. In contrast, hem1Δ cells lacking hri1+ (hri1Δ) exhibit poor eIF2α phosphorylation under the same stress conditions. When ALA-starved hem1Δ hri1Δ cells are subsequently transferred to a medium supplemented with exogenous hemin, they exhibit impaired growth compared to ALA-starved hem1Δ cells expressing the endogenous hri1+ allele or hem1Δ hri1Δ hri2Δ gcn2Δ cells expressing functional hri1+ and hri1+-GFP alleles. Consistent with its role as a heme-sensing eIF2α kinase, further analysis by absorbance spectroscopy demonstrates that Hri1 binds to hemin, with an equilibrium dissociation constant (KD) of 0.11 µM. In contrast, a truncated form of Hri1 (from residues 1–185) fails to interact with hemin. Taken together, these findings provide the first report of a fungal eIF2α kinase being activated in response to stress directly linked to a defect in heme homeostasis.