Tokumoto Umechiyo, Nomura Shinobu, Minami Yoshiko, Mihara Hisaaki, Kato Shin-Ichiro, Kurihara Tatsuo, Esaki Nobuyoshi, Kanazawa Hiroshi, Matsubara Hiroshi, Takahashi Yasuhiro
Department of Biology, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
J Biochem. 2002 May;131(5):713-9. doi: 10.1093/oxfordjournals.jbchem.a003156.
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.
铁硫（Fe-S）簇的组装由复杂的机制介导，在大肠杆菌中，该机制由iscRSUA-hscBA-fdx-ORF3基因簇编码。在此，我们展示了该机制中各组分之间的蛋白质-蛋白质相互作用网络。我们构建了各组分的（His）6标签版本，并鉴定了它们与通过镍亲和柱从大肠杆菌提取物中共纯化的相互作用伙伴。使用双杂交系统在酵母细胞中进一步检测了确定的蛋白质对之间的直接关联。与之前的体外结合和动力学实验一致，观察到IscS与IscU、IscU与HscB、IscU与HscA以及HscB与HscA之间的相互作用。此外，我们还鉴定了IscS与Fdx、IscS与ORF3、IscA与HscA以及HscA与Fdx之间先前未报道的相互作用。我们还通过定点突变分析结合双杂交系统发现，IscU中的两个半胱氨酸残基对于与HscB结合至关重要，但与IscS结合则不然。尽管各组分的各种组合存在复杂的相互作用网络，但除了推测的IscU-IscS-ORF3寡聚形式外，我们的实验中未观察到异源多聚体复合物。因此，IscS、IscU、IscA、HscB、HscA、Fdx和ORF3蛋白之间的顺序缔合和解离可能是Fe-S簇组装中的关键过程。
