In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation

Abstract

FeS-clusters are utilized by numerous proteins within several biological pathways that are essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway. In Saccharomyces cerevisiae, de novo FeS-cluster formation is accomplished through coordinated assembly with the substrates iron and sulfur by the scaffold assembly protein “Isu1”. Sulfur for cluster assembly is provided by cysteine desulfurase “Nfs1”, a protein that works in union with its accessory protein “Isd11”. Frataxin “Yfh1” helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of sulfur and Fe(II) to Isu1, or more likely through a combination of these and other possible roles. In vitro studies on the yeast ISC machinery have been limited, however, due to the inherent instability of recombinant Isu1. Isu1 is a molecule prone to degradation and aggregation. To circumvent Isu1 instability, we have replaced yeast Isu1 with the fly ortholog to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our laboratory previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to the yeast ortholog. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrate its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has similar physical properties similar to that of yeast Isu1. It functions as a stable dimer with similar Fe(II) affinity and ability to form two 2Fe-2S clusters as the yeast dimer. The fIscU and yeast ISC proteins are compatible in vitro; addition of Yfh1 to Nfs1-Isd11 increases the rate of FeS-cluster formation on fIscU to a similar extent observed with Isu1. Finally, fIscU expressed in mitochondria of a yeast strain lacking Isu1 (and its paralog Isu2) is able to completely reverse the deletion phenotypes. These results demonstrate fIscU can functionally replace yeast Isu1 and it can serve as a powerful tool for exploring molecular details within the yeast ISC pathway.

Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe–S cluster assembly centers

“…The UV-visible absorption spectra recorded at the end of each reaction (Fig. S13C and D, ESI † ) had features similar to those recently shown for the holo forms of yeast Isu1 and fly IscU, with absorbance inflections between 420 and 450 nm, 96 which is characteristic of [2Fe–2S]-bound IscU-type proteins. 60 , 97 The [2Fe–2S] yield estimated at A = 426 nm with an extinction coefficient ε = 8–11 mM –1 cm –1 ( ref.…”

“… 32 , 105 However, beyond molecular details of the Yfh1–Isu1 interaction and modeling, 32 , 41 the putative [Nfs1–Isd11–Isu1–Yfh1] 2 complex has remained largely uncharacterized structurally and as it pertains in particular to the path for iron delivery to Isu1. 96 Consequently, an integrated mechanism for a concerted delivery of sulfur and iron for cluster assembly is lacking.…”

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

“…The cytosolic electron donors cytokine-induced apoptosis inhibitor-1 (CIAPIN1) and glutaredoxins Grx3 and Grx4 have been identified (Figure 1 ). Only three genes involved in Fe-S biosynthesis have been individually studied in flies: (i) Drosophila frataxin whose exact biochemical function is still unclear (Cañizares et al, 2000 ; Anderson et al, 2005 , 2008 ; Llorens et al, 2007 ; Kondapalli et al, 2008 ; Runko et al, 2008 ; Navarro et al, 2010 , 2011 , 2015 ; Shidara and Hollenbeck, 2010 ; Soriano et al, 2013 , 2016 ; Tricoire et al, 2014 ; Calap-Quintana et al, 2015 ; Palandri et al, 2015 ; Chen et al, 2016b ; Edenharter et al, 2017 ), (ii) IscU that encodes a protein assembly platform for Fe-S cluster biosynthesis (Dzul et al, 2017 ), and iii) Hsc20 encoding one of two chaperones that mobilize the Fe-S cluster from IscU to downstream Fe-S proteins in the mitochondria (Uhrigshardt et al, 2013 ).…”

“…Two Drosophila Fe-S cluster assembly proteins have been purified to date, frataxin (Kondapalli et al, 2008 ) and IscU (Dzul et al, 2017 ). We characterized the core mitochondrial protein complex involved in Fe-S cluster assembly in flies (Marelja, 2013 ).…”

Iron–sulfur cluster biogenesis and trafficking in mitochondria

“…How iron gains access to the active site remains enigmatic. Apo-Isu1 does not appear to bind iron at the active site with significant affinity (58), suggesting that iron may be actively delivered to the ISC complex. Yfh1 and its human homologue frataxin can bind ferrous iron via acidic residues on the N-terminal ␣-helix, but concrete in vivo evidence of Yfh1 supplying iron is still lacking (15,59).…”