Abstract
In this study, we generated a Schizosaccharomyces pombe strain deficient in heme and siderophore biosynthesis, as well as in reductive iron uptake. Using this hem1Δ sib1Δ sib2Δ fio1Δ fip1Δ mutant strain, we identified Hhy1, a novel protein required for optimal growth when cells rely on exogenous hemin as their sole source of heme. The expression of Hhy1 is upregulated under low-iron conditions and downregulated when iron levels increase. Fluorescence microscopy analysis shows that the Hhy1-GFP fluorescent signal mirrors hhy1(+) mRNA expression, being primarily detected in the cytoplasm of iron-starved hem1Δ sib1Δ sib2Δ fio1Δ fip1Δ cells. Further monitoring of intracellular heme or its analog zinc mesoporphyrin IX revealed that hem1Δ sib1Δ sib2Δ fio1Δ fip1Δ cells lacking Hhy1 accumulate these molecules more persistently than cells expressing Hhy1. Consistent with a role for Hhy1 in heme homeostasis, hhy1Δ cells exhibit reduced heme-dependent activity of the catalase Ctt1. Coimmunoprecipitation and bimolecular fluorescence complementation experiments show that Hhy1 interacts with Ctt1. Absorbance spectroscopy and hemin-agarose pull-down assays demonstrate that Hhy1 binds hemin, with an equilibrium dissociation constant of 1.09 μM. Taken together, these findings indicate that the iron-regulated cytoplasmic hemoprotein Hhy1 interacts with Ctt1, promoting its full activation against oxidative stress under hemin-dependent growth conditions.