Epistasis between synonymous and nonsynonymous mutations in Dictyostelium discoideum ammonium transporter amtA drives functional complementation in Saccharomyces cerevisiae

Abstract

AbstractRole of Horizontal Gene Transfer (HGT) in evolution transcends across the three domains of life. Ammonium transporters are present in all species and therefore offer an excellent paradigm to study protein evolution following HGT. While investigating HGT through complementation assay, we observed that synonymous and nonsynonymous mutations follow an epistastic relationship. As a proxy for HGT, we attempted to complement a mep1mep2mep3Δ strain of S. cerevisiae (triple deletion strain) which cannot grow on ammonium as a sole nitrogen source below a concentration of 3 mM, with amtA of D. discoideum. As the wild type amtA did not complement, we isolated two mutant derivatives of amtA that complemented the triple deletion strain of S. cerevisiae. amtA M1 bears three nonsynonymous and two synonymous substitutions and these substitutions are necessary for its functionality. amtA M2 bears two nonsynonymous and one synonymous substitution, all of which are necessary for functionality. These mutants were then studied at phenotypic, cell biological, and biochemical level. Interestingly, AmtA M1 transports ammonium but does not confer toxicity to methylamine while AmtA M2 transports ammonium as well as confers methylamine toxicity, demonstrating functional diversification. Based on the results presented, we suggest that protein evolution cannot be fathomed by studying nonsynonymous and synonymous substitutions separately. This is because, protein evolution entails an interaction between synonymous and nonsynonymous substitution, which seems to have gone unnoticed thus far. Above observations have significant implications in various facets of biological processes and are discussed in detail.HighlightsAmmonium transporters (Amts) from bacteria to humans complement in yeastAmtA of D. discoideum does not complement yeast defective for ammonium uptakeSynonymous & nonsynonymous mutations are essential for AmtA functionality in yeastConformational differences underlie functionality & functional diversificationProtein evolution entails interaction of synonymous & nonsynonymous mutations