title: Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli.
abstract: The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.
authors: Department of Biology, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
authors: Umechiyo
authors: U
authors: Tokumoto
authors: Shinobu
authors: S
authors: Nomura
authors: Yoshiko
authors: Y
authors: Minami
authors: Hisaaki
authors: H
authors: Mihara
authors: Shin-Ichiro
authors: S
authors: Kato
authors: Tatsuo
authors: T
authors: Kurihara
authors: Nobuyoshi
authors: N
authors: Esaki
authors: Hiroshi
authors: H
authors: Kanazawa
authors: Hiroshi
authors: H
authors: Matsubara
authors: Yasuhiro
authors: Y
authors: Takahashi
mesh_terms: D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006632:Histamine / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D006632:Histamine; D006026:Glycoside Hydrolases; C023112:exo-beta-D-galactofuranosidase; D001616:beta-Galactosidase; D003545:Cysteine