The Final Maturation State of β-actin Involves N-terminal Acetylation by NAA80, not N-terminal Arginylation by ATE1.

Adrian Drazic, Evy Timmerman, Ulrike Kajan, Michaël Marie, Sylvia Varland, Francis Impens, Kris Gevaert, Thomas Arnesen

Journal of molecular biology 2022 Jan 30

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Actin is a hallmark protein of the cytoskeleton in eukaryotic cells, affecting a range of cellular functions. Actin dynamics is regulated through a myriad of actin-binding proteins and post-translational modifications. The mammalian actin family consists of six different isoforms, which vary slightly in their N-terminal (Nt) sequences. During and after synthesis, actins undergo an intricate Nt-processing that yields mature actin isoforms. The ubiquitously expressed cytoplasmic β-actin is Nt-acetylated by N-alpha acetyltransferase 80 (NAA80) yielding the Nt-sequence Ac-DDDI-. In addition, β-actin was also reported to be Nt-arginylated by arginyltransferase 1 (ATE1) after further peptidase-mediated processing, yielding RDDI-. To characterize in detail the Nt-processing of actin, we used state-of-the-art proteomics. To estimate the relative cellular levels of Nt-modified proteoforms of actin, we employed NAA80-lacking cells, in which actin was not Nt-acetylated. We found that targeted proteomics is superior to a commercially available antibody previously used to analyze Nt-arginylation of β-actin. Significantly, despite the use of sensitive mass spectrometry-based techniques, we could not confirm the existence of the previously claimed Nt-arginylated β-actin (RDDI-) in either wildtype or NAA80-lacking cells. A very minor level of Nt-arginylation of the initially cleaved β-actin (DDDI-) could be identified, but only in NAA80-lacking cells, not in wildtype cells. We also identified small fractions of cleaved and unmodified β-actin (DDI-) as well as cleaved and Nt-acetylated β-actin (Ac-DDI-). In sum, we show that the multi-step Nt-maturation of β-actin is terminated by NAA80, which Nt-acetylates the exposed Nt-Asp residues, in the virtual absence of previously claimed Nt-arginylation. Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Citation

Adrian Drazic, Evy Timmerman, Ulrike Kajan, Michaël Marie, Sylvia Varland, Francis Impens, Kris Gevaert, Thomas Arnesen. The Final Maturation State of β-actin Involves N-terminal Acetylation by NAA80, not N-terminal Arginylation by ATE1. Journal of molecular biology. 2022 Jan 30;434(2):167397