Abstract
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.
Highlights
L-cysteine desulfurases are pyridoxal phosphate (PLP)-dependent enzymes that use L-cysteine as substrate to produce L-alanine and a protein-bound persulfide [1,2]
NFS1 forms a complex with ISD11, a 10 kDa protein that functions as stabilizer of NFS1 [7,8]
In order to confirm the cytosolic role of NFS1 for molybdenum cofactor (Moco) biosynthesis in vivo, we showed the cytosolic localization of NFS1 and performed interaction studies between NFS1 and MOCS3 in HeLa cells
Summary
L-cysteine desulfurases are pyridoxal phosphate (PLP)-dependent enzymes that use L-cysteine as substrate to produce L-alanine and a protein-bound persulfide [1,2]. NFS1 forms a complex with ISD11, a 10 kDa protein that functions as stabilizer of NFS1 [7,8]. While L-cysteine desulfurases are highly conserved throughout all kingdoms of life, ISD11 is only found in eukaryotes, suggesting a unique and novel function of the protein for this class of organisms [9]. NFS1/ ISD11 binds to the scaffold protein ISCU forming the ternary ISCU/NFS1/ISD11 complex [10]. This complex provides the platform for binding of frataxin, a protein of the core ISC assembly machinery regulating the activity of the ternary complex [11]. Since binding sites for iron have been detected on its surface, frataxin has been proposed to be the iron donor for the ISC assembly machinery in former studies, this role is not clear [12,13,14]
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