Nuclear Localization of Yeast Nfs1p Is Required for Cell Survival

Abstract

Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.The NifS protein is found in various organisms and is involved in iron-sulfur protein biosynthesis. In the diazotroph Azotobacter vinelandii, the nifS gene was first identified as a member of the nif operon that plays an essential role in the biosynthesis of nitrogenase (1). Extensive biochemical analyses of A. vinelandii NifS reveal that it is a PLP 1 -containing enzyme exhibiting desulfurase activity that produces elemental sulfur and L-alanine from L-cysteine (2). These findings suggest that the physiological function of NifS is to supply inorganic sulfur for the assembly of the iron-sulfur cluster in nitrogenase (3, 4).Many eukaryotic NifS homologues have been identified, of which the Saccharomyces cerevisiae NifS homologue, Nfs1p, is the best characterized (5-9). The nfs1 null mutant is lethal (5, 9), and Nfs1p is localized mostly to the mitochondrial matrix (7-9). Mitochondrial Nfs1p mediates the assembly of the ironsulfur cluster of both mitochondrial and cytosolic iron-sulfur proteins (6, 8) and also regulates mitochondrial and cytosolic iron homeostasis (9). Kolman and Söll (5) also show that a mutation in the NFS1 gene suppressed a certain tRNA-splicing mutant (5), although the role of Nfs1p in the process of tRNA splicing, which is thought to take place in the nucleus, remains unclear.Mouse and human counterparts to Nfs1p (mNfs1 and hNfs1, respectively) have also been identified (7, 10). We showed that mNfs1 was found mainly in the mitochondrial matrix (7). The full-length hNfs1 protein also possesses a mitochondrial-targeting presequence and has been shown to be localized to mitochondria. Different sized hNfs1 proteins from a single transcript have also been detected in the cytosolic and nuclear fractions (10), but the physiological significance of these extramitochondrial hNfs1 proteins has not been elucidated.The Escherichia coli NifS homologue, IscS, is a multi-functional enzyme that possesses important physiological functions not only in iron-sulfur cluster assembly (11, 12) but also in sulfur transfer to some intracellular compounds (13-18). These results suggest that a fraction of Nfs1p may have some essential function other than iron-sulfur cluster assembly, possibly in a different subcellular location. In...

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