Figure 5: Functional reconstitution of the microsporidian CIA scaffold complex.

(a) Yeast complementation assay. Functionality of the T. hominis CIA components ThNar1, ThCfd1, ThCia1 or ThNbp35 was tested by their ability to rescue the growth defect of respective regulatable Gal-CIA yeast mutants on glucose-containing minimal medium (s.d.). Indicated cells were transformed with plasmid p416-MET25 containing either no gene (p416), the respective yeast (Sc) or the homologous T. hominis (Th) CIA genes. Gal-CFD1 cells were additionally transformed with p415-MET25 to allow co-production of both ThCfd1 and ThNbp35 (right). Cells were depleted of the respective nuclear-encoded CIA proteins by growth on s.d. medium for the indicated times at 30 °C. Serial tenfold dilutions were spotted onto agar plates containing either SGal (minimal medium plus galactose) or s.d. medium, and growth was continued for 2 days at 30 °C. None of the T. hominis genes improved growth of the yeast CIA protein-depleted cells. (b) T. hominis ThCfd1 and His-tagged ThNbp35 were co-expressed in E. coli and co-purified as a complex by affinity chromatography (insight, right). Fe/S clusters were chemically reconstituted on the ThCfd1–HisThNbp35 complex resulting in a dark-brown protein solution (inset, left). Ultraviolet–vis spectroscopy revealed an absorption peak around 420 nm indicative of the formation of [4Fe–4S] clusters on the complex. (c) The presence of [4Fe–4S] clusters was confirmed by X-band EPR spectroscopy of a reduced sample (40 μM, treated with 0.2 mM Na-dithionite) recorded at 10 K. Experimental conditions: frequency 9.6359 GHz, power 1 mW, modulation 0.75 mT/100 kHz. The numbers are the principal g values obtained by simulation.