Department of Molecular Enzymology, Institute of Biochemistry, University of Potsdam, Potsdam, Germany.
PLoS One. 2013 Apr 12;8(4):e60869. doi: 10.1371/journal.pone.0060869. Print 2013.
In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Förster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.
在人类中，L-半胱氨酸脱硫酶 NFS1 在线粒体铁硫簇生物合成和线粒体及细胞质 tRNA 的硫修饰中起着至关重要的作用。我们之前已经证明，纯化的 NFS1 能够将硫转移到参与钼辅因子生物合成和 tRNA 硫修饰的细胞质蛋白 MOCS3 的 C 末端结构域。然而，目前还没有直接证据表明 NFS1 和 MOCS3 在人细胞的细胞质中相互作用。在这里，我们使用Förster 共振能量转移和分裂 EGFP 系统提供了直接证据，表明 NFS1 和 MOCS3 在人细胞的细胞质中相互作用。通过对细胞分级和使用共聚焦荧光显微镜进行定位研究的免疫检测，证实了 NFS1 和 MOCS3 在细胞质中的共定位。纯化的 NFS1 被用于重建 Neurospora crassa nit-1 突变体中缺失的钼酶活性，这进一步证明 NFS1 是真核生物中 Moco 生物合成的硫供体。
