The cytosolic iron sulfur cluster assembly (CIA) pathway is responsible for the maturation of cytosolic and nuclear iron-sulfur proteins. These proteins play vital roles in numerous essential processes critical for cell growth and division, including DNA replication and repair, transcription, and translation. Although recent studies have linked CIA dysfunction or dysregulation to genome instability and an increased mutation rate, the underlying molecular mechanisms have not been elucidated due, in part, to our limited understanding of this essential biochemical pathway. One major unsolved problem is how the CIA system recognizes its &gt;40 substrates so that their cofactors can be inserted. The CIA targeting complex (CTC), comprising the Met18, Cia1 and Cia2 subunits in <i>Saccharomyces cerevisiae,</i> is proposed to perform target identification during the final step of the CIA pathway. However, it is not known which of the CTC subunits are critical for identifying apo-FeS proteins and what primary or tertiary structural elements of the targets are responsible for recruitment of the CTC. Here we report that a C-terminal aromatic residue is both necessary and sufficient for association of several different FeS proteins to the targeting complex. Bioinformatic analysis reveals that up to 25% CTC binding proteins and CIA substrates terminate in a conserved aromatic residue, most commonly tryptophan. Using an in vitro affinity copurification approach, we have defined the primary structural elements critical for CTC binding. Current efforts are focused on determining what residues or regions of the targeting complex are necessary for identification of this CIA targeting motif.

Discovery of a motif that is necessary and sufficient for metallocluster delivery by the cytosolic iron sulfur cluster assembly pathway

ACS Fall 2021

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