Title
In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation.
Author
Stephen Dzul, Agostinho Rocha, Swati Rabat, Ashoka Kandegedara, April Kusowski, Jayashree Pain, Anjaneyulu Murari, Debkumar Pain, Andrew Dancis, Timothy Stemmler
Year
2016
Journal
Metallomics
Abstract
FeS-clusters are utilized by numerous proteins within several biological pathways essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway which has has been extensively characterized within Saccharomyces cerevisiae. In yeast, de novo FeS-cluster formation is accomplished through coordinated assembly of substrates iron and sulfur on 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 Fe(II) to Isu1, or more likely through a combination of roles. In vitro studies on the yeast ISC have been limited, however, due to the inherent instability of recombinant Isu1, a molecule prone to degradation and aggregation. To circumvent Isu instability, we have replaced yeast Isu1 with the fly ortholog in order to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our lab previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to Yfh1. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrated its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has similar physical properties to yeast Isu1, functions as a stable dimer with similar Fe(II) affinity and ability to form two 2Fe-2S clusters as the yeast dimer both. 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 as observed with yeast 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, both in vivo and in vitro.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Ligand binding, Coordination chemistry, Kinetics, Biochemistry