Subcellular compartmentalization of human Nfu, an iron-sulfur cluster scaffold protein, and its ability to assemble a [4Fe-4S] cluster

Abstract

Iron-sulfur (Fe-S) clusters serve as cofactors in many proteins that have important redox, catalytic, and regulatory functions. In bacteria, biogenesis of Fe-S clusters is mediated by multiple gene products encoded by the isc and nif operons. In particular, genetic and biochemical studies suggest that IscU, Nfu, and IscA function as scaffold proteins for assembly and delivery of rudimentary Fe-S clusters to target proteins. Here we report the characterization of human Nfu. A combination of biochemical and spectroscopic techniques, including UV-visible absorption and 57Fe Mössbauer spectroscopies, have been used to investigate the ability of purified human Nfu to assemble Fe-S clusters. The results suggest that Nfu can assemble approximately one labile [4Fe-4S] cluster per two Nfu monomers, and support the proposal that Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. Analyses of genomic DNA, transcripts, and translation products indicate that alternative splicing of a common pre-mRNA results in synthesis of two Nfu isoforms with distinct subcellular localizations. Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. These results, together with previous reports of subcellular distributions of isoforms of human IscS and IscU in mitochondria, cytosol, and nucleus suggest that the Fe-S cluster assembly machineries are compartmentalized in higher eukaryotes.

Fig. 1.

Alternative splicing of nfu pre-mRNA results in two isoforms with different translation initiation sites. (A) Predicted sequences of the two human Nfu isoforms. The boxed region indicates the coding sequences. Note that HIRIP5 was previously reported to initiate at AUG3. The asterisk indicates an in-flame stop codon present only in isoform II transcript. (B) Schematic mechanism for the use of alternative 5′ splice sites in exon I of nfu to generate two isoforms with different translational initiation sites (AUG1 or AUG2). (C) Gel electrophoretic analysis of RT-PCR DNA fragments obtained by using two different primer sets. Arrows indicate fragments amplified from isoform I, while asterisks indicate fragments amplified from isoform II.

Fig. 2.

Nfu isoform I is localized to mitochondria, whereas isoform II is localized to the cytosol and nucleus. (A) Schematic diagram of the nfu constructs used for in vitro translation and COS-7 cell transfection. (B) COS-7 cells transfected with constructs containing either isoform I or isoform II were stained with rabbit polyclonal Abs to Nfu (α-NFU 4333) and were analyzed by immunofluorescence microscopy. Arrows denote transfected cells. Mitochondria were stained with MitoTracker 633, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

Fig. 3.

Endogenous Nfu proteins are present in the cytosol, mitochondria, and nucleus of RD4 cells. (A) Western blot analysis of subcellular fractions of RD4 cell lysates by using α-Nfu 4333 and α-Nfu(MAP) 4337. Analysis of a nuclear protein (Oct-1) and a mitochondrial protein (Mn-SOD) are included for comparison. (B) Western blot analysis of RD4 triton lysate (lane 1) using α-Nfu 4333. In vitro translation products of pcDNA-nfu isoform I, pcDNA-nfu isoform II, and a control construct, pPoly(A)-luciferase, were shown for comparison. (C) RD4 cells were pulse-labeled with [³⁵S]methionine and chased at the specified times. Immunoprecipitation of the triton lysates withα-Nfu 4333 indicated a two-step processing of Nfu isoform I. (D) Immunoprecipitation of endogenous Nfu in RD4 lysates, compared with immunoprecipitation of the in vitro translation products of nfu isoform I and II. As expected, in vitro translation of isoform I gives rise to a major protein of ≈28 kDa, whereas isoform II gives rise to a protein of ≈26 kDa. The shorter product of isoform II (≈22 kDa) may be the products of in vitro translation initiated at ATG3 (Fig. 1A).

Fig. 4.

UV-visible absorption spectra of Nfu (43 μM) before (A) and after (B) in vitro cluster assembly.

Fig. 5.

Mössbauer spectra of human Nfu (430μM) after in vitro assembly of Fe–S cluster. The spectra (hatched marks) were recorded at 4.2 K in a field of 50 mT (A) or 6 T (B) applied parallel to the γ-radiation. The solid lines overlaid with the experimental spectra are theoretical simulations using two overlapping quadrupole doublets of the following parameters and assuming diamagnetism: δ = 0.47 mm/s, ΔE_Q = 1.35 mm/s, andη = 0.5 for doublet 1, andδ = 0.47 mm/s, ΔE_Q = 1.09 mm/s, and η = 0.7 for doublet 2.