On the Role of Nbp35 in Cytosolic Iron-Sulfur Assembly
thesisposted on 2016-10-29, 00:00 authored by Kelly A. Gay
Iron-sulfur clusters are versatile protein cofactors, essential to virtually all forms of life. Apbc/Nbp35 P-Loop NTPases are required for iron-sulfur cluster biogenesis in almost all organisms, and are proposed to function as a scaffold for assembly of nascent cluster. Nbp35 functions in complex with the homologous NTPase Cfd1 in yeast and higher eukaryotes. Previous investigations demonstrated a requirement for the nucleotide domain in Nbp35 and Cfd1. However, the roles of nucleotide binding and hydrolysis have remained unclear. Therefore these investigations were designed to determine the roles of nucleotide binding and hydrolysis by Nbp35 in cluster biogenesis. NTPases such as Nbp35 share significant architectural and mechanistic similarities. Therefore conserved residues within cognate NTPase motifs were targeted by directed mutagenesis, and the effects of these mutations on known Nbp35 functions were assessed in yeast. Targeted motifs included the P-Loop and Switch I, which play established roles in nucleotide binding, hydrolysis, or the signaling of nucleotide status. Most mutations to these motifs were lethal, underscoring their importance to Nbp35 function. Although stably expressed and competent for complex formation, these mutants were defective for the acquisition of iron, as assessed by in vivo radio-labeling. These results suggest that Nbp35 activities may be required for self-maturation, prior to assembly of labile cluster. These investigations targeted two additional residues, conserved in homologous NTPases. Although outside established NTPase motifs, these residues map to a conserved region, and are required for hydrolysis and allosteric signaling in homologous proteins. These mutants were partially functional, but displayed defects in cluster assembly, including the acquisition of iron. Preliminary results suggest these residues may be required for assembly and/or transfer of labile cluster by Nbp35. These studies identify a conserved helix, required in homologous NTPases, and extend a requirement for this region to Nbp35, suggesting this region may constitute a novel motif in this family. Collectively, these investigations demonstrate that Nbp35 activities are required for self-maturation AND assembly of labile cluster for transfer, and suggest that the mechanism of cytosolic iron-sulfur assembly may be more complex than previously thought.