The L-Cysteine Desulfurase NFS1 Is Localized in the Cytosol where it Provides the Sulfur for Molybdenum Cofactor Biosynthesis in Humans

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.

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

“…The function of NFS1 in the cytosol was described to be a sulfur donor for MOCS3, a dual function protein that interacts with URM1 for tRNA thiolation and with MOCS2A for the biosynthesis of Moco (22,53). Thus, to characterize the role of TUM1 in humans, we analyzed the interaction of both isoforms with MOCS3 and NFS1.…”

“…To date, the principal function of NFS1 in the cytosol has been to perform sulfur transfer to MOCS3 for Moco biosynthesis and the thiolation of cytoplasmic tRNAs (53). In this compartment, TUM1-Iso1 interacts with both MOCS3 and NFS1.…”

Shared function and moonlighting proteins in molybdenum cofactor biosynthesis

“…Further, the sulfur delivery pathway to MOCS3 involves the l-cysteine desulfurase NFS1 in the cytosol (Marelja et al, 2013). In eukaryotes, however, NFS1 was described to be mainly localized in the mitochondria where it is involved in the assembly of FeS clusters (Biederbick et al, 2006) (Figure 4).…”

“…Genes and the encoded proteins were named in humans as MOCS (molybdenum cofactor synthesis) (Reiss et al, 1998b;Reiss, 2000). Additionally, at least two further proteins are involved in Moco biosynthesis: the l-cysteine desulfurase NFS1 (Marelja et al, 2013) and the human Moco sulfurase HMCS (Peretz et al, 2007). In comparison to the components involved in Moco biosynthesis identified in bacteria, homologous proteins in humans are present, however, these consist either of two fused domains or alternatively fusions at the gene level are present, which by alternative splicing produce the active protein subunits.…”

“…Last but not least, NFS1 is a l-cysteine desulfurase with a main cellular localization in mitochondria where its primary role is dedicated to FeS cluster assembly (Biederbick et al, 2006). However, NFS1 was also identified to be localized in the cytosol where it interacts with MOCS3 and thereby provides the sulfur both for Moco biosynthesis and tRNA thiolation (Marelja et al, 2013) (Table 1).…”