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1.
Biochemistry ; 58(12): 1587-1595, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30785732

RESUMO

The cytosolic iron sulfur cluster assembly (CIA) scaffold biosynthesizes iron sulfur cluster cofactors for enzymes residing in the cytosol and the nucleus. In fungi and animals, it comprises two homologous ATPases, called Nbp35 and Cfd1 in yeast, which can form homodimeric and heterodimeric complexes. Both proteins are required for CIA function, but their individual roles are not well understood. Here we investigate the nucleotide affinity of each form of the scaffold for ATP and ADP to reveal any differences that could shed light on the functions of the different oligomeric forms of the protein or any distinct roles of the individual subunits. All forms of the CIA scaffold are specific for adenosine nucleotides and not guanosine nucleotides. Although the Cfd1 homodimer has no detectable ATPase activity, it binds ATP with an affinity comparable to that of the hydrolysis competent forms, Nbp352 and Nbp35-Cfd1. Titrations to determine the number of nucleotide binding sites combined with site-directed mutagenesis demonstrate that the nucleotide must bind to the Cfd1 subunit of the heterodimer before it can bind to Nbp35 and that the Cfd1 subunit is hydrolysis competent when bound to Nbp35 in the heterodimer. Altogether, our work reveals the distinct roles of the Nbp35 and Cfd1 subunits in their heterodimeric complex. Cfd1 controls nucleotide binding, and the Nbp35 subunit is required to activate nucleotide hydrolysis.


Assuntos
Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Domínio Catalítico , Proteínas de Ligação ao GTP/genética , Proteínas Ferro-Enxofre/genética , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética
2.
Biochemistry ; 57(16): 2349-2358, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-28539047

RESUMO

The cytosolic iron-sulfur cluster assembly (CIA) system assembles iron-sulfur (FeS) cluster cofactors and inserts them into >20 apoprotein targets residing in the cytosol and nucleus. Three CIA proteins, called Cia1, Cia2, and Met18 in yeast, form the targeting complex responsible for apo-target recognition. There is little information about the structure of this complex or its mechanism of CIA substrate recognition. Herein, we exploit affinity co-purification and size exclusion chromatography to determine the subunit connectivity and stoichiometry of the CIA targeting complex. We conclude that Cia2 is the organizing center of the targeting complex, which contains one Met18, two Cia1, and four Cia2 polypeptides. To probe target recognition specificity, we utilize the CIA substrates Leu1 and Rad3 as well as the Escherichia coli FeS-binding transcription factor FNR (fumerate nitrate reductase). We demonstrate that both of the yeast CIA substrates are recognized, whereas the bacterial protein is not. Thus, while the targeting complex exhibits flexible target recognition in vitro, it cannot promiscuously recognize any FeS protein. Additionally, we demonstrate that the full CIA targeting complex is required to stably bind Leu1 in vitro, whereas the Met18-Cia2 subcomplex is sufficient to recognize Rad3. Together, these results allow us to propose a unifying model for the architecture of this highly conserved complex and demonstrate what component or subcomplexes are vital for target identification.


Assuntos
Núcleo Celular/química , Citosol/química , Proteínas Ferro-Enxofre/química , Mapas de Interação de Proteínas/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , DNA Helicases/química , DNA Helicases/genética , Hidroliases/química , Hidroliases/genética , Proteínas Ferro-Enxofre/genética , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética
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