Abstract
Actin is one of the most abundant eukaryotic cytoskeletal polymer-forming proteins, which, in the filamentous form, regulates a number of physiological processes, ranging from cell division and migration to development and tissue function. Actins have different post-translational modifications (PTMs) in different organisms, including methionine, alanine, aspartate and glutamate N-acetylation, N-arginylation and the methylation of the histidine at residue 73 (His-73), with different organisms displaying a distinct signature of PTMs. Currently, methods are not available to produce actin isoforms with an organism-specific PTM profile. Here, we report the Pick-ya actin method, a method to express actin isoforms from any eukaryote with its own key characteristic PTM pattern. We achieve this using a synthetic biology strategy in a yeast strain that expresses, one, actin isoforms with the desired N-end via ubiquitin fusion and, two, mammalian enzymes that promote acetylation and methylation. Pick-ya actin should greatly facilitate biochemical, structural and physiological studies of the actin cytoskeleton and its PTMs.
Highlights
Actin is a highly conserved eukaryotic cytoskeletal protein, which polymerizes into double-stranded, dynamic polar filaments that exhibit continuous polymerization and depolymerization
When the four purified actin fusions were subjected to mass spectrometry (MS), we found that the majority of the N-termini in β actin contained an aspartate residue, whereas a large number of acetylated aspartate (Ac-Asp)containing peptides were detected when NAA80 was co-expressed in these cells (Fig. 2B,D; Fig. S1A)
In this study, we have substantially improved upon our previous work (Hatano et al, 2018) on expression and purification of heterologous actin isoforms in P. pastoris
Summary
Actin is a highly conserved eukaryotic cytoskeletal protein, which polymerizes into double-stranded, dynamic polar filaments that exhibit continuous polymerization and depolymerization. A variety of eukaryotes have evolved diverged actin isoforms and orthologues, which assemble into a variety of molecular machineries executing mechanical tasks, including cell division and cell migration. How actins undergo defined molecular structural changes facilitating polymerization and depolymerization, and how they carry out distinct functions at different cellular locations and in different organisms and tissues is not well understood. Some of the known modifications of actin include: (1) methionine N-acetylation in yeast; (2) alanine N-acetylation in Arabidopsis thaliana (Bienvenut et al, 2012); Centre for Mechanochemical Cell Biology and Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK. *These authors contributed to this work
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