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

• Published in: Molecular microbiology Vol. 115; no. 4; pp. 699 - 722
• Main Authors: Normant, Vincent, Brault, Ariane, Avino, Mariano, Mourer, Thierry, Vahsen, Tobias, Beaudoin, Jude, Labbé, Simon
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.04.2021; Wiley
• Subjects: Biochemistry; Biochemistry, Molecular Biology; Biosynthesis; Biotinylation; Cell surface; Cellular Biology; fission yeast; Fluorescence; Heme; Heme proteins; Hemin; hemoprotein; Hydrogen peroxide; Iron; Life Sciences; major facilitator transporter; Mass spectrometry; Mass spectroscopy; Membrane proteins; Membranes; Microbiology and Parasitology; Molecular biology; Mycology; Peroxidase; Peroxiredoxin; Proteins; Schizosaccharomyces pombe; Subcellular Processes; hemoprotein; peroxiredoxin; major facilitator transporter; fission yeast; heme
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.14638

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 proximity‐dependent 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 H2O2. 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. Under conditions where Schizosaccharomyces pombe cannot synthesize heme de novo, this organism uses two distinct uptake systems to acquire exogenous heme. One system requires the cell surface transporter Str3 (blue). When iron and oxygen levels are low, the cytosolic peroxiredoxin Tpx1 (green) interacts with Str3 in a hemin‐dependent manner, revealing a new role as cytosolic scavenger for Tpx1. Site‐directed mutagenesis revealed that conserved Cys‐Pro (CP) motifs of Tpx1 are required for its hemin‐binding ability.