Identification of two Cryptococcus neoformans heme transporters involved in Fhb1-mediated nitrosative stress protection in a fission yeast model

In Schizosaccharomyces pombe and Cryptococcus neoformans, the flavohemoglobins (flavoHbs) that protect these yeasts against nitrosative stress are Yhb1 and Fhb1, respectively. Although heme is an essential cofactor for flavoHb activity, the molecular circuitry governing the acquisition of exogenous heme by flavoHbs remains largely unknown. Because S. pombe is a powerful model organism for studying heme biology, Fhb1 was heterologously expressed in S. pombe mutant cells lacking Yhb1. Fhb1 expression functionally complements yhb1Δ cells that are defective in nitric oxide (˙NO) detoxification. Moreover, this Fhb1-mediated complementation requires the S. pombe heme transporter Str3 or the heterologous coexpression of two previously uncharacterized C. neoformans transporters, CnStr1 and CnStr3. Consistent with this finding, heterologous expression of CnStr1 and CnStr3 rescues the heme-dependent growth defect of S. pombe cells that are deficient in heme biosynthesis and lack endogenous heme uptake systems when hemin is provided as the sole heme source. Coimmunoprecipitation assays further demonstrate interactions between Fhb1 and S. pombe Str3 or C. neoformans Str3-like proteins when coexpressed in fission yeast cells. In C. neoformans, strains lacking CnStr1 and CnStr3 exhibit a heme-dependent growth defect in the absence of the cell-surface hemophore Cig1. Similar to the C. neoformans fhb1Δ mutant, a cig1Δ Cnstr1Δ Cnstr3Δ triple mutant fails to protect cells from nitrosative stress. Collectively, these findings reveal functional conservation between S. pombe Yhb1 and C. neoformans Fhb1 in their ability to acquire exogenous heme required for activation as defenders against nitrosative stress.