The N‑Terminus of Iron–Sulfur Cluster Assembly Factor ISD11 Is Crucial for Subcellular Targeting and Interaction with l‑Cysteine Desulfurase NFS1

Assembly of iron–sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by l-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondrial targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved “LYR” motif is essential for the role of ISD11 in forming a stable and active complex with NFS1

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

<div><p>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 <i>Neurospora crassa nit-1</i> mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.</p> </div

Immunodetection of NFS1 and MOCS3 after subcellular fractionation of HeLa cells.

<p>Total protein extracts (T), cytosol (C), mitochondria (M), and nucleus (N) were prepared separately from 80–90% confluent HeLa cells to avoid cross contaminations of the compartments. Proteins of each cellular fraction were analyzed by immunoblotting using the following antibodies: anti-NFS1 (<i>top panel</i>), anti-γ-actin as cytosolic marker control (<i>second panel</i>), anti-laminB1 as nuclear marker (<i>third panel</i>), anti-ABCB7 as mitochondrial inner membrane marker (<i>fourth panel</i>), anti-MOCS3 as cytosolic marker (<i>fifth panel</i>), and anti-citrate synthase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060869#pone.0060869-Kispal1" target="_blank">[6]</a> as mitochondrial matrix marker (<i>bottom</i>).</p

Analysis of protein-protein interactions between MOCS3 and NFS1Δ1-55 by SPR measurements.

a<p>Proteins were immobilized via amine coupling.</p>b<p>Proteins were injected using KINJECT protocol, injecting samples in a concentration range of 0.3–10 µM. Flow cells were regenerated by injection of 20 mM HCl.</p>c<p>K_D mean values with standard deviation were obtained from 3 independent measurements after global fitting procedures for 1∶1 binding for each measurement.</p>d<p>−, binding not calculated.</p

Fluorescent microscopy of EYFP/ECFP fusion proteins expressed in HeLa cells.

<p>EYFP (<i>green pseudocolor</i>) and ECFP-tagged (<i>red pseudocolor</i>) proteins were analyzed in HeLa cells for subcellular localization and co-localization (the “<i>merge</i>” row resulted in a <i>yellow color</i> when colocalization occurred) by fluorescence confocal microscopy. In addition, a line profile plot (<i>right</i>) shows the pixel intensities of EYFP and ECFP along an arrow (distance in µm) presented in the “merge” figure. <i>A,</i> NFS1-EYFP and ISD11-ECFP (mitochondrial localization); <i>B</i>, NFS1-EYFP (cytosolic localization) and ISD11-ECFP (nuclear localization); <i>C</i>, EYFP-NFS1Δ1-55 and ISD11-ECFP; <i>D</i>, <i>E</i> NFS1-EYFP and ECFP-MOCS3; <i>F</i>, EYFP-NFS1Δ1-55 and ECFP-MOCS3. The figures of <i>panel E</i> are a <i>close-up</i> figure of an indicated area (box) in the figures of <i>panel D</i>. Scale bars, 20 µm (except <i>E,</i> which is 2 µm).</p

Quantification of the ECFP donor fluorescence lifetime (τ₁) by time-resolved FRET in HeLa cells.

<p>After expression of ECFP and EYFP-tagged proteins in HeLa cells, the ECFP donor lifetime was determined by FRET measurements and the long lifetime component of the bi-exponentially decaying ECFP is shown. <i>A</i>, ECFP donor controls, showing expression of ECFP-EYFP fusion proteins, ECFP and EYFP, and ECFP-MOCS3 and EYFP-MOCS2A. <i>B</i>, ECFP donor lifetime of MOCS3-MoeBD-ECFP, ECFP-MOCS3-RLD, ECFP-MOCS3 and ECFP-MOCS3/EYFP. <i>C</i>, ECFP donor lifetime of MOCS3-MoeBD-ECFP, ECFP-MOCS3-RLD, and ECFP-MOCS3 fusion in presence of EYFP-NFS1Δ1-55 is shown. For each value N = 30–42 cells were measured (displayed as mean ± standard deviation).</p

Analysis of NFS1 and MOCS3 interactions by using the split-EGFP system in HeLa cells.

<p>Subcellular EGFP assembly of different split-EGFP fusion proteins was analyzed in HeLa cells by confocal fluorescent microscopy. The following fusion proteins were expressed after cotransfection (assembly of EGFP^1–157 and EGFP^158–238 resulted in a <i>green pseudocolor</i>): <i>A</i>, ISD11-EGFP^1–157 and NFS1-EGFP^158–238; <i>B</i>, MOCS3-EGFP^1–157 and NFS1-EGFP^158–238; <i>C</i>, MOCS3-EGFP^1–157 and NFS1Δ1-55-EGFP^158–238; <i>D</i>, MOCS3-EGFP^1–157 and MOCS3-EGFP^158–238; <i>E</i>, NFS1-EGFP^1–157 and NFS1-EGFP^158–238; <i>F</i>, ISD11-EGFP^1–157 and NFS1Δ1-55-EGFP^158–238. Mitochondria of HeLa cells were visualized with MitoTracker® DeepRed (<i>red</i>) or the nuclei were visualized with DAPI stain (<i>magenta</i>). Merged pictures are shown right (either resulting in a <i>yellow</i> or <i>white color</i>). Scale bars, 20 µm; scale bars in the insets, 2 µm.</p

Fluorescent microscopy of EYFP/ECFP fusion proteins expressed in HeLa cells.

<p>EYFP (<i>green pseudocolor</i>) and ECFP-tagged (<i>red pseudocolor</i>) proteins were analyzed in HeLa cells for subcellular localization and co-localization (the “<i>merge</i>” row resulted in a <i>yellow color</i> when colocalization occured) by fluorescence confocal microscopy. In addition, a line profile plot (<i>right</i>) shows the pixel intensities of EYFP and ECFP along an arrow (distance in µm) presented in the “merge” figure. <i>A,</i> EYFP-MOCS2A and ECFP-MOCS3; <i>B</i>, EYFP-MOCS2A and NFS1-ECFP; <i>C</i>, EYFP-MOCS2A and ECFP-NFS1Δ1-55; <i>D</i>, EYFP and ECFP. Scale bars, 20 µm.</p

Bacterial plasmids used in this study.

<p>Bacterial plasmids used in this study.</p

Reconstitution of nitrate reductase activity in <i>N. crassa nit-1</i> extracts using different sulfur donors.

<p>All reconstitution mixtures contained 30 µl freshly prepared <i>N. crassa nit-1</i> extracts, 10 µl 0.5 M sodium molybdate, and 10 µM of MOCS3 and/or NFS1Δ1-55 or an excess of isolated Moco and 10 µM active <i>E. coli</i> MPT synthase (positive control). As sulfur source either 1 mM L-cysteine or 1 mM sodium thiosulfate was added. The reaction mixtures were incubated for 30 min at room temperature, then another 20 min for the reaction of the reconstituted nitrate reductase, and finally the reaction was stopped and the produced nitrite was determined at 540 nm. <i>n.d</i>., not detectable.</p