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A Method to Purify Actin Isoforms with Bespoke Key Post-Translational

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A Method to Purify Actin Isoforms with Bespoke Key Post-Translational © 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs241406. doi:10.1242/jcs.241406 TOOLS AND RESOURCES Pick-ya actin – a method to purify actin isoforms with bespoke key post-translational modifications Tomoyuki Hatano*,‡, Lavanya Sivashanmugam*, Andrejus Suchenko, Hamdi Hussain and Mohan K. Balasubramanian‡ ABSTRACT (3) aspartate and glutamate N-acetylation in animals and amoeba, Actin is one of the most abundant eukaryotic cytoskeletal polymer- mediated by the N-acetyl transferase NAA80 (Drazic et al., 2018; forming proteins, which, in the filamentous form, regulates a number Goris et al., 2018); (4) N-arginylation in mammals (Karakozova of physiological processes, ranging from cell division and migration et al., 2006); and (5) His-73 methylation in animals and amoeba, to development and tissue function. Actins have different post- mediated by the histidine methyl transferase SETD3 (Kwiatkowski translational modifications (PTMs) in different organisms, including et al., 2018; Wilkinson et al., 2019). Thus, a precise biochemical and methionine, alanine, aspartate and glutamate N-acetylation, biophysical understanding of actin function in various organisms and N-arginylation and the methylation of the histidine at residue 73 tissues depends on the ability to produce actin isoforms with (His-73), with different organisms displaying a distinct signature of organism-specific signature PTMs. The current methods for actin PTMs. Currently, methods are not available to produce actin isoforms purification either constitutively introduce aspartate and glutamate with an organism-specific PTM profile. Here, we report the Pick-ya N-acetylation and His-73 methylation (purification from rabbit or Dictyostelium actin method, a method to express actin isoforms from any eukaryote chicken muscle, Sf9-insect cell cultures, and human with its own key characteristic PTM pattern. We achieve this using a platelets) or do not introduce either of the above-mentioned Saccharomyces cerevisiae synthetic biology strategy in a yeast strain that expresses, one, actin modifications (purification from and Pichia pastoris isoforms with the desired N-end via ubiquitin fusion and, two, ) (Hatano et al., 2018; Müller et al., 2013; Noguchi mammalian enzymes that promote acetylation and methylation. et al., 2007; Ohki et al., 2009). P. pastoris Pick-ya actin should greatly facilitate biochemical, structural and Given that recombinant actin expressed in is neither physiological studies of the actin cytoskeleton and its PTMs. N-acetylated on aspartate and glutamate residues nor His-73 methylated, we reasoned that further engineering of P. pastoris KEY WORDS: Pichia pastoris, Actin cytoskeleton, Post-translational may provide a means to express and purify actin isoforms with modification, Recombinant actin distinct organism-specific PTMs. Here, we express NAA80 and SETD3 either singly or together in P. pastoris. This, combined with INTRODUCTION our previously described ubiquitin fusion approach (Hatano et al., Actin is a highly conserved eukaryotic cytoskeletal protein, which 2018), has led to the development of the Pick-ya actin method, polymerizes into double-stranded, dynamic polar filaments that which facilitates production of actin isoforms with organism- exhibit continuous polymerization and depolymerization. A variety specific PTM profiles. of eukaryotes have evolved diverged actin isoforms and orthologues, which assemble into a variety of molecular machineries executing RESULTS mechanical tasks, including cell division and cell migration. How Fig. 1 shows key PTMs observed in actin from mammals, yeast and actins undergo defined molecular structural changes facilitating the plant A. thaliana. Mammalian actin can be isolated with one of polymerization and depolymerization, and how they carry out seven different modifications at the N-terminus, which are mediated distinct functions at different cellular locations and in different by a combination of aminopeptidases, NAA80 acetyl transferase organisms and tissues is not well understood. The understanding of (Drazic et al., 2018; Goris et al., 2018) and ATE1 arginyl-tRNA- actin function in different organisms and tissues is further protein transferase (Karakozova et al., 2006). Furthermore, human complicated by the vast array of PTMs, which differ among actin is methylated on His-73 by the SETD3 methyl transferase different organisms. Some of the known modifications of actin (Kwiatkowski et al., 2018; Wilkinson et al., 2019). A. thaliana actin include: (1) methionine N-acetylation in yeast; (2) alanine is N-terminally acetylated at alanine and is only weakly (9%) N-acetylation in Arabidopsis thaliana (Bienvenut et al., 2012); methylated on His-74, the equivalent residue to mammalian and yeast His-73 (Wilkinson et al., 2019). Finally, yeast actin is Centre for Mechanochemical Cell Biology and Division of Biomedical Sciences, acetylated on the initiator methionine and is not methylated on His- Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK. 73 (Kalhor et al., 1999). We systematically established yeast strains *These authors contributed equally to this work that introduced one or more of the above-mentioned modifications. ‡Authors for correspondence ([email protected]; Towards this end, we cloned human NAA80 acetyl transferase and [email protected]) human SETD3 methyl transferase in P. pastoris expression plasmids, which are as described in the subsequent sections. M.K.B., 0000-0002-1292-8602 This is an Open Access article distributed under the terms of the Creative Commons Attribution Expression in P. pastoris and purification of N-acetylated License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, β γ distribution and reproduction in any medium provided that the original work is properly attributed. human and actin Human β- and γ-actin expressed in P. pastoris are not processed at Received 6 November 2019; Accepted 20 December 2019 the N-terminus, as a result methionine is retained at the N-terminus Journal of Cell Science 1 TOOLS AND RESOURCES Journal of Cell Science (2020) 133, jcs241406. doi:10.1242/jcs.241406 Fig. 1. Post-translational modification of actin. Divergent post-translational modifications on actin. Shown are N-terminal peptide sequences of nine actins (six from H. sapiens, one from S. cerevisiae, and three from A. thaliana), and the observed modifications at the N-terminus. The right column summarizes whether His-73 methylation is observed in these organisms. and is acetylated by native acetyl transferases (Hatano et al., 2018). His-73 on β- and γ-actin expressed in P. pastoris (Fig. 3D,E). The canonical N-acetylation occurs on an aspartate residue on These experiments established that human β- and γ-actins can be β-actin and a glutamate residue on γ-actin. To facilitate generation of efficiently methylated by SETD3 in P. pastoris. β- and γ-actin with aspartate or glutamate at the N-terminus, we expressed a DNA molecule (Fig. 2A) encoding a translational Expression in P. pastoris and purification of N-acetylated and fusion of ubiquitin with the actin gene lacking the initiator His-73 methylated human β-actin, and arginylated and His-73 methionine followed by sequences encoding human thymosin β4 methylated γ-actin (Tβ4) and the eight consecutive histidine amino acid residues Next, we sought to purify dually modified human β-actin with (8His), which facilitates one-step affinity purification using nickel- N-acetylation and His-73 methylation. Towards this goal, we NTA resin (Hatano et al., 2018; Huang et al., 2016; Kijima et al., employed a ubiquitin-fusion strategy as described in Fig. 2A. In P. 2016; Noguchi et al., 2007). In this strain, we introduced an pastoris cells expressing the Ub–D-actin-Tβ4–8His fusion, we integrative plasmid vector backbone or a plasmid expressing human expressed NAA80 and SETD3. Actin was purified sequentially by NAA80, which promotes actin N-acetylation. The translational nickel affinity chromatography, release from Tβ4 and 8His by fusion was purified as described previously using an affinity- chymotrypsin treatment, and a cycle of polymerization and binding approach (Hatano et al., 2018). When the four purified actin depolymerization (see Materials and Methods for full details of fusions were subjected to mass spectrometry (MS), we found that the purification). Fig. 4A shows results from a size exclusion the majority of the N-termini in β actin contained an aspartate column, in which monomeric actin was obtained in the peak residue, whereas a large number of acetylated aspartate (Ac-Asp)- fractions. Mass spectrometric analysis of the purified actin containing peptides were detected when NAA80 was co-expressed confirmed that it was both N-acetylated and His-73 methylated in these cells (Fig. 2B,D; Fig. S1A). Similarly, the majority of γ- (Fig. S2), as described in Figs 2 and 3. actin contained a glutamate residue at the N-terminus in control Next, we tested whether the purified N-acetylated and His-73 cells, whereas it contained acetylated glutamate (Ac-Glu) at the methylated human β-actin was capable of undergoing N-terminus when NAA80 was co-expressed (Fig. 2C,E). Evaluation polymerization. Towards this goal, we incubated the purified of the number of non-acetylated versus acetylated N-terminal human actin with a trace amount of S. cerevisiae actin that was peptides from β- and γ-actin clearly demonstrated that NAA80 fluorescently labelled by conjugation
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