Actin is a major eukaryotic cytoskeletal protein that supports a large spectrum of cellular processes. In different organisms and tissues, actin may be post-translationally modified through acetylation, arginylation and/or methylation to serve different functions. However, so far, there has been no means to produce and subsequently study these different actin isoforms in vitro. Now, Tomoyuki Hatano, Mohan Balasubramanian and colleagues (Hatano et al., 2020) develop Pick-ya actin, a strategy to generate actin isoforms with organism-specific post-translational modifications. This method involves the expression of recombinant actin in Pichia pastoris, which does not carry either N-acetylated aspartate and glutamate or methylated histidine 73. However, because recombinant human actin is not processed to remove the N-terminal methionine in P. pastoris, the authors express actin as a fusion with ubiquitin, which is removed by an endogenous ubiquitin-specific protease. By co-expressing the human NAA80 acetyltransferase or the SETD3 methyltransferase with actin, the authors can generate actin molecules that are acetylated or methylated at specific residues. Moreover, the authors also show that expression of SETD3 causes methylation of N-arginylated actin. Thus, Pick-ya actin represents a novel strategy to produce recombinant actin isoforms with targeted post-translational modifications, which will greatly facilitate the study of these isoforms in the future.
