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. 2023:2620:209-217.
doi: 10.1007/978-1-0716-2942-0_23.

Reconstitution of the Arginyltransferase (ATE1) Iron-Sulfur Cluster

Verna Van  1 , Aaron T Smith  2
Affiliations

Reconstitution of the Arginyltransferase (ATE1) Iron-Sulfur Cluster

Verna Van et al. Methods Mol Biol. 2023.

Abstract

As global regulators of eukaryotic homeostasis, arginyltransferases (ATE1s) have essential functions within the cell. Thus, the regulation of ATE1 is paramount. It was previously postulated that ATE1 was a hemoprotein and that heme was an operative cofactor responsible for enzymatic regulation and inactivation. However, we have recently shown that ATE1 instead binds an iron-sulfur ([Fe-S]) cluster that appears to function as an oxygen sensor to regulate ATE1 activity. As this cofactor is oxygen-sensitive, purification of ATE1 in the presence of O2 results in cluster decomposition and loss. Here, we describe an anoxic chemical reconstitution protocol to assemble the [Fe-S] cluster cofactor in Saccharomyces cerevisiae ATE1 (ScATE1) and Mus musculus ATE1 isoform 1 (MmATE1-1).

Keywords: Anoxic reconstitution; Arginylation; Arginyltransferases; Chemical reconstitution; Ferrozine assay; Iron-sulfur clusters.

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References

    1. Van V, Smith AT (2020) ATE1-mediated post-translational Arginylation is an essential regulator of eukaryotic cellular homeostasis. ACS Chem Biol 15(12):3073–3085 - DOI - PubMed - PMC
    1. Kashina A (2014) Protein arginylation, a global biological regulator that targets actin cytoskeleton and the muscle. Anat Rec (Hoboken) 297:1630–1636 - DOI - PubMed
    1. Kwon YT, Kashina AS, Davydov IV, Hu R-G, An JY, Seo JW, Du F, Varshavsky A (2002) An essential role of N-terminal Arginylation in cardiovascular development. Science 297:96–99 - DOI - PubMed
    1. Hu R-G, Sheng J, Qi X, Xu Z, Takahashi TT, Varshavsky A (2005) The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators. Nature 437:981–986 - DOI - PubMed
    1. Moorthy BT, Jiang C, Patel DM, Ban Y, O’Shea CR, Kumar A, Yuan T, Birnbaum MD, Gomes AV, Chen X, Fontanesi F, Lampidis TJ, Barrientos A, Zhang F (2022) The evolutionarily conserved arginyltransferase 1 mediates a pVHL-independent oxygen-sensing pathway in mammalian cells. Dev Cell 57:654–669.e9 - DOI - PubMed - PMC

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