Human Nbp35 Is Essential for both Cytosolic Iron-Sulfur Protein Assembly and Iron Homeostasis

Abstract

The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

“…Estimating the protein amounts of several Fe/S proteins is also informative with respect to a cell's capacity to form the metallo-cofactor (25,26). In line with the aforementioned results, the levels of mitochondrial aconitase, cytosolic aconitase (IRP1) and SDH were all decreased under Fdx2 depletion (Fig.…”

“…4B). For IRP1 the decrease is readily explained by the common observation that apo-IRP1 lacking its Fe/S cluster is more prone to degradation (26,35). Because IRP2 protein levels are negatively regulated by the iron availability in the cytosol (34), our results suggest that cytosolic iron was diminished.…”

Analysis of iron–sulfur protein maturation in eukaryotes

“…Finally, cell density affects the cellular iron status, which in turn can complicate Fe/S protein activity measurements, in particular those of cytosolic aconitase/IRP1 (ref. 23).…”

“…This requirement does not pose a limitation in experiments with yeast cells, as their growth is only mildly affected at low iron concentrations and the frequent use of plasmids urges the use of defined (minimal) media. For radiolabeling of human cell lines, however, the depletion of iron severely decreases cell growth 23 , and an efficient radiolabeling procedure has not yet been described.…”

“…In fact, seven mammalian relatives of yeast ISC and CIA proteins have been experimentally studied to date and shown to serve a similar function in the cell as their yeast counterparts [17][18][19][20][21][22][23] . The crucial importance of Fe/S proteins and their assembly machineries has been recognized by virtue of the association of lesions in ISC machinery genes with three human diseases (for a review, see refs.…”