Fig 5. Fbx15 is required for nuclear localization of SsnF.

(A) Fluorescence microscopy of SsnF-GFP in wild type or Δfbx15 background revealed that SsnF-GFP was localized to the periphery of the nuclei in Δfbx15, whereas it was inside the nuclei in wild type. (B) SsnF-GFP localization in wild type Fbx15-RFP, unphosphorylated Fbx15[S468|9A]-RFP or phosphorylated Fbx15[S469D]-RFP in backgrounds before and after H₂O₂-treatment. Under normal growth conditions SsnF is nuclear in the presence of either the wild type or the phosphate mimicking Fbx15[S469D] variant, whereas SsnF-GFP accumulates at the nuclear envelope in presence of the non-phosphorylatable Fbx15[S468|9A]. Upon oxidative stress, the dephosphorylation of wild type Fbx15 leads to similar subpopulations of SsnF like the unphosphorylated Fbx15[S468|9A]. In phosphorylated Fbx15[S469D] SsnF enters the nucleus even after H₂O₂-treatment. (C) Growth tests of A. fumigatus strains expressing either wild type fbx15::rfp (AfGB98), rfp tagged phosphovariants of fbx15 (AfGB101 and AfGB102) or corresponding rfp-tagged versions of fbx15 that lack the F-box domain (AfGB125, AfGB126 and AfGB127). 5 x 10³ conidia were spotted on minimal medium (MM) plate, supplemented with 2 mM H₂O₂ for providing oxidative stress, and grown for four days at 37°C.