Functional analysis of yeast L-cysteine desulfurase Nfs1p, an specific sulfan-sulfur supplier to various micromolecules

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The IscS protein is a pyridoxal-phosphate(PLP)-containing cysteine desulfurase involved in iron-sulfur cluster (ISC) biogenesis. In prokaryotes, IscS is also involved in various metabolic functions including thio-modification of tRNA. By contrast, the eukaryotic orthologue of IscS (Nfs1) has thus far only been shown to be functional in mitochondrial ISC biogenesis. We demonstrate here that yeast Nfs1p is also required for the post-transcriptional thio-modification of both mitochondrial (mt-) and cytoplasmic (cy-) tRNAs in vivo. Depletion of Nfs1p resulted in an immediate impairment of 2-thio-modification of 5-carboxymethylaminomethyl-2-thiouridine (cmnm^5s^2U) at the wobble positions of mt-tRNA^<Lys>_<UUU> and mt-tRNA^<Gln>_<UUG-> In addition, we observed a severe reduction in the 2-thio-modification of 5-methoxycarbonylmethyl-2-thiouridine (mcm^5s^2U) of cy-tRNA^<Lys2>_<UUU> and cy-tRNA^<Glu3>_<UUC> although the effect was somewhat delayed compared to that seen in mt-tRNAs. Mass spectrometric analysis revealed an increase in 5-methoxycarbonylmethyluridine (mcm^5U) concomitant with the decrease in mcm^5s^2U in cy-tRNAs that were prepared from Nfs1p-depleted cells. These results suggest that Nfs1p is involved in the 2-thio-modifications of both cmnm^5s^2U in mt-tRNAs and also mcm^5s^2U in cy-tRNAs.