PMC

Purified Nbp35p is an Fe/S protein. UV-vis spectrum of N-terminally His-tagged Nbp35p (1.9 mg/ml/50 mM Tris·HCl, pH 8/1 μM 5′-deazaflavin) before (OX) and after (DAF) photoreduction by irradiation for 3 min with a commercial slide projector. The contribution of oxidized 5′-deazaflavin to the spectrum before photoreduction has been subtracted. Inset shows the results of colorimetric iron and acid-labile sulfide determinations for five independent samples.

Overexpression of NBP35 and NAR1 inhibits growth of Cfd1p-depleted Gal-CFD1 cells. (A) Gal-NBP35 and Gal-CFD1 cells were transformed with the high-copy plasmids p424-NBP35 and p426-CFD1-HA or empty vectors (-) to overexpress NBP35 and CFD1 under control of the TDH3 promoter as indicated. Tenfold serial dilutions of cells were cultivated on solid minimal media supplemented with galactose or glucose at 30°C twice for 3 days. (B) Gal-CFD1 cells transformed with high copy vectors encoding the indicated genes were grown as in A.

Nbp35p is required for maturation of Fe/S proteins in the cytosol and nucleus but not in mitochondria. (A) Gal-NBP35 cells overproducing HA-tagged (HA) versions of Rli1p and Nar1p were cultivated in iron-poor minimal media containing galactose (Gal) or glucose (Glc). Cells were labeled with ⁵⁵Fe and lysed, and the radiolabeled proteins were immunoprecipitated by using anti-HA or anti-Leu1p antibodies. Coimmunoprecipitated ⁵⁵Fe was quantified by liquid scintillation counting. (B and C) Wild-type (WT+) and Gal-NBP35 cells overproducing a HA-tagged (HA) version of Ntg2p (B) or mitochondrial Bio2p (C), and wild-type cells carrying vector p426GPD (WT-; B) were radiolabeled with ⁵⁵Fe under permissive (Gal) and repressive (Glc) conditions. Iron binding to Ntg2p and Bio2p was determined by immunoprecipitation as described above. Insets show the immunostaining of indicated proteins in the cell extracts.

Fe/S cluster association at the N terminus of Nbp35p requires a functional mitochondrial ISC machinery. (A) Wild-type cells overproducing TAP-tagged versions of Nbp35p (+) or Nbp35pΔ1-52 (Δ1-52) from vector p426-NBP35-TAP and control cells carrying plasmid p426GPD (-) were grown in iron-poor minimal medium supplemented with either galactose (Gal) or glucose (Glc) for 16 h. Cells were radiolabeled with ⁵⁵Fe for 4 h, and cell lysates were prepared. TAP-tagged Nbp35p proteins were immunoprecipitated from the lysates with IgG, and the amount of coimmunoprecipitated ⁵⁵Fe was quantified by liquid scintillation counting. (B) Gal-NFS1 and Gal-YAH1 cells overproducing Nbp35p-TAP were grown as in A to allow or repress, respectively, NFS1 and YAH1 expression. Subsequently, ⁵⁵Fe binding to Nbp35p-TAP was investigated as described above. (C) Gal-ATM1 cells overproducing Nbp35p-TAP were treated as cells in A, and ⁵⁵Fe binding to Nbp35p-TAP and the endogenous Leu1p was determined. Insets show immunostaining of the indicated proteins in the cell extracts.