Hemeprotein Tpx1 interacts with cell‐surface heme transporter Str3 in Schizosaccharomyces pombe

Abstract

Str3 is a transmembrane protein that mediates low-affinity heme uptake in Schizosaccharomyces pombe. Under iron-limiting conditions, Str3 remains at the cell surface in the presence of increasing hemin concentrations. Using a proximitydependent biotinylation approach coupled to mass spectrometry and coimmunoprecipitation assays, we report that the peroxiredoxin Tpx1 is a binding partner of Str3.Under microaerobic conditions, cells deficient in heme biosynthesis and lacking the heme receptor Shu1 exhibit poor hemin-dependent growth in the absence of Tpx1.Analysis of membrane protein preparations from iron-starved hem1Δ shu1Δ str3Δ tpx1Δ cells coexpressing Str3-GFP and TAP-Tpx1 showed that TAP-Tpx1 is enriched in membrane protein fractions in response to hemin. Bimolecular fluorescence complementation assays brought additional evidence that an interaction between Tpx1 and Str3 occurs at the plasma membrane. Results showed that Tpx1 exhibits an equilibrium constant value of 0.26 μM for hemin. The association of Tpx1 with hemin protects hemin from degradation by H 2 O 2 . The peroxidase activity of hemin is lowered when it is bound to Tpx1. Taken together, these results revealed that Tpx1 is a novel interacting partner of Str3. Our data are the first example of an interaction between a cytoplasmic heme-binding protein and a cell-surface heme transporter.

A novel role of the fission yeast sulfiredoxin Srx1 in heme acquisition

“…In our previous study (Normant et al, 2021), we reported the enrichment of S. pombe Tpx1 in the membrane fraction along with Str3 when cells are compelled to utilize the Str3-dependent hemin uptake system for growth. Under these conditions, some Tpx1 proteins form a heteroprotein complex with Str3, which is detectable through pull-down assays (Normant et al, 2021). Similarly, in this study, coimmunoprecipitation experiments showed the interaction between Srx1-Myc 12 and Str3-GFP proteins in iron-starved cells grown in the presence of hemin.…”

“…It is known that Srx s interact with 2-Cys Prx s (Jönsson et al, 2008(Jönsson et al, , 2009. Previous studies have shown that S. pombe 2-Cys peroxiredoxin Tpx1 and its human ortholog Prx1 can directly interact with hemin (Normant et al, 2021;Watanabe et al, 2017). Therefore, we F I G U R E 3 Analysis of Srx1-GFP protein steady-state levels and its localization as a function of iron availability.…”

“…Yeast liquid proliferation assays in ALA-free medium supplemented with hemin were performed as described previously (Normant et al, 2021). Under microaerobic growth conditions, fission yeast cells were grown in culture jars with BD GasPak EZ (BD Diagnostic System, Sparks, MD).…”

“…In the case of coimmunoprecipitation of MBP-Tpx1/Srx1-His 6 and Str3-GFP/Srx1-Myc 12 heteroprotein complexes, assays were conducted in the presence of monoclonal antibodies against MBP and GFP, coupled to protein G-Sepharose 4 Fast Flow beads, respectively. The resulting immunoprecipitated proteins were dissociated from the beads using the method described previously (Normant et al, 2021). Following elution, the attached protein complexes were analyzed by immunoblot assays using the corresponding antibody pairs: anti-MBP and anti-His 6 or anti-GFP and anti-Myc.…”

“…The controlled uptake of heme by cells is crucial due to its potentially toxic nature. In the context of the Str3‐dependent heme acquisition pathway, Str3 remains stable at the plasma membrane even in the presence of increasing hemin concentrations (Normant et al, 2021). This suggests the involvement of cellular factors in heme mobilization during or after its transport across the plasma membrane.…”