The eukaryotic P loop NTPase Nbp35: an essential component of the cytosolic and nuclear iron-sulfur protein assembly machinery

Abstract

Soluble P loop NTPases represent a large protein family and are involved in diverse cellular functions. Here, we functionally characterized the first member of the Mrp/Nbp35 subbranch of this family, the essential Nbp35p of Saccharomyces cerevisiae. The protein resides in the cytosol and nucleus and carries an Fe/S cluster at its N terminus. Assembly of the Fe/S cluster requires the mitochondrial Fe/S cluster (ISC)-assembly and -export machineries. Depletion of Nbp35p strongly impairs the activity of the cytosolic Fe/S protein, isopropylmalate isomerase (Leu1p), whereas mitochondrial Fe/S enzymes are unaffected. Moreover, defects in the de novo maturation of various cytosolic and nuclear Fe/S proteins were observed in the absence of Nbp35p, demonstrating the functional involvement of Nbp35p in the biogenesis of extramitochondrial Fe/S proteins. Furthermore, Nbp35p genetically interacts with the closely similar P loop NTPase, Cfd1p, and the hydrogenase-like Nar1p, both of which were recently shown to perform a crucial function in cytosolic and nuclear Fe/S protein biogenesis. Hence, our study suggests that eukaryotic Nbp35 NTPases function in Fe/S protein maturation. The findings provide strong evidence for the existence of a highly conserved and essential machinery dedicated to assembling cytosolic and nuclear Fe/S proteins.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.