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

酵母 L-半胱氨酸脱硫酶 Nfs1p（各种小分子的特定硫磺供应者）的功能分析

基本信息

批准号：
15570127
负责人：
NAKAI Yumi
金额：
$ 2.05万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570127/
关键词：
Nfs1p thio-modification tRNA iron-sulfur cluster biogenesis mitochondrial cytoplasmic サイトゾル硫黄原子 IscS

项目摘要

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.

IscS蛋白是一种含有磷酸吡哆醛（PLP）的半胱氨酸脱硫酶，参与铁硫簇（ISC）的生物合成。在原核生物中，IscS还参与多种代谢功能，包括tRNA的硫代修饰。相比之下，IscS（Nfs 1）的真核直向同源物迄今为止仅被证明在线粒体ISC生物发生中起作用。我们在这里证明，酵母Nfs 1 p也需要在体内的线粒体（mt-）和细胞质（cy-）的tRNA的转录后硫代修饰。Nfs 1 p的缺失导致mt-tRNA^ _和mt-tRNA^ _摆动位置上的5-羧甲基氨基甲基-2-硫代尿苷的2-硫代修饰（cmnm^5s^2U）立即受损<Lys>。<UUU><Gln><UUG->此外，我们观察到cy-tRNA ^_和cy-tRNA^ _的5-甲氧羰基甲基-2-硫代尿苷的2-硫代修饰（mcm^5s^2U）严重减少<Lys2><UUU><Glu3>，<UUC>尽管与mt-tRNA相比，这种影响有些延迟。质谱分析显示，在从Nfs 1 p缺失的细胞中制备的cy-tRNA中，5-甲氧羰基甲基尿苷（mcm^5U）增加，同时mcm^5s^2U减少。这些结果表明，Nfs 1 p参与了mt-tRNA中cmnm^5s^2U和cy-tRNA中mcm^5s^2U的2-硫代修饰。