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

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 (3-actin is Nt-acetylated by N-alpha acetyltransferase 80 (NAA80) yielding the Nt-sequence Ac-DDDI-. In addition, (3-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 (3-actin. Significantly, despite the use of sensitive mass spectrometry-based techniques, we could not confirm the existence of the previously claimed Nt-arginylated (3-actin (RDDI-) in either wildtype or NAA80-lacking cells. A very minor level of Ntarginylation of the initially cleaved (3-actin (DDDI-) could be identified, but only in NAA80-lacking cells, not in wildtype cells. We also identified small fractions of cleaved and unmodified (3-actin (DDI-) as well as cleaved and Nt-acetylated (3-actin (Ac-DDI-). In sum, we show that the multi-step Nt-maturation of (3-actin is terminated by NAA80, which Nt-acetylates the exposed Nt-Asp residues, in the virtual absence of previously claimed Nt-arginylation.(c) 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).