Role of Human Mitochondrial Nfs1 in Cytosolic Iron-Sulfur Protein Biogenesis and Iron Regulation

Abstract

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a complex process involving more than 20 components. So far, functional investigations have mainly been performed in Saccharomyces cerevisiae. Here, we have analyzed the role of the human cysteine desulfurase Nfs1 (huNfs1), which serves as a sulfur donor in biogenesis. The protein is located predominantly in mitochondria, but small amounts are present in the cytosol/nucleus. huNfs1 was depleted efficiently in HeLa cells by a small interfering RNA (siRNA) approach, resulting in a drastic growth retardation and striking morphological changes of mitochondria. The activities of both mitochondrial and cytosolic Fe/S proteins were strongly impaired, demonstrating that huNfs1 performs an essential function in Fe/S protein biogenesis in human cells. Expression of murine Nfs1 (muNfs1) in huNfs1-depleted cells restored both growth and Fe/S protein activities to wild-type levels, indicating the specificity of the siRNA depletion approach. No complementation of the growth retardation was observed, when muNfs1 was synthesized without its mitochondrial presequence. This extramitochondrial muNfs1 did not support maintenance of Fe/S protein activities, neither in the cytosol nor in mitochondria. In conclusion, our study shows that the essential huNfs1 is required inside mitochondria for efficient maturation of cellular Fe/S proteins. The results have implications for the regulation of iron homeostasis by cytosolic iron regulatory protein 1.

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

“…Mitochondria of Fdx2-deficient, but not of Fdx1-deficient cells exhibited a substantial loss of cristae membranes (Fig. S2), consistent with the severe effects on respiratory chain function upon Fdx2 depletion (23,24).…”

Analysis of iron–sulfur protein maturation in eukaryotes

“…This procedure substantially diminishes background values and in many cases is crucial for detection of low levels of activity (e.g., for cytosolic aconitase in human cells 19 ). If necessary, marker enzymes can be artificially introduced into an organism or cellular compartment by ectopic expression.…”

“…Likewise, Table 3 provides representative specific enzyme activities in galactose-grown wild-type cells. Appropriate non-Fe/S enzymes should be measured in parallel as controls 10,19,68 . Figure 3 shows typical results for an in vivo 55 Fe incorporation experiment in yeast.…”

“…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.…”

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

“…The biogenesis of Fe-S clusters is performed by two main multi-protein machineries, the ISC (Iron-Sulfur Cluster) machinery localized in mitochondria and the CIA (Cytosolic Iron-sulfur cluster Assembly) machinery localized in the cytosol 16 . The ISC machinery provides Fe-S clusters for mitochondrial proteins and also synthesizes an unknown compound that is exported to the cytosol and required for assembly of Fe-S clusters by the CIA machinery [17][18][19] .…”