Densi Asha, Iyer Revathi S, Bhat Paike Jayadeva
Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India.
Microbiol Spectr. 2023 Feb 22;11(2):e0384722. doi: 10.1128/spectrum.03847-22.
Ammonium transporters are present in all three domains of life. They have undergone extensive horizontal gene transfer (HGT), gene duplication, and functional diversification and therefore offer an excellent paradigm to study protein evolution. We attempted to complement a ΔΔΔ strain of Saccharomyces cerevisiae (triple-deletion strain), which otherwise cannot grow on ammonium as a sole nitrogen source at concentrations of <3 mM, with of Dictyostelium discoideum, an orthologue of S. cerevisiae . We observed that did not complement the triple-deletion strain of S. cerevisiae for growth on low-ammonium medium. We isolated two mutant derivatives of ( and ) from a PCR-generated mutant plasmid library that complemented the triple-deletion strain of S. cerevisiae. bears three nonsynonymous and two synonymous substitutions, which are necessary for its functionality. bears two nonsynonymous substitutions and one synonymous substitution, all of which are necessary for functionality. Interestingly, AmtA M1 transports ammonium but does not confer methylamine toxicity, while AmtA M2 transports ammonium and confers methylamine toxicity, demonstrating functional diversification. Preliminary biochemical analyses indicated that the mutants differ in their conformations as well as their mechanisms of ammonium transport. These intriguing results clearly point out that protein evolution cannot be fathomed by studying nonsynonymous and synonymous substitutions in isolation. The above-described observations have significant implications for various facets of biological processes and are discussed in detail. Functional diversification following gene duplication is one of the major driving forces of protein evolution. While the role of nonsynonymous substitutions in the functional diversification of proteins is well recognized, knowledge of the role of synonymous substitutions in protein evolution is in its infancy. Using functional complementation, we isolated two functional alleles of the D. discoideum ammonium transporter gene (), which otherwise does not function in S. cerevisiae as an ammonium transporters. One of them is an ammonium transporter, while the other is an ammonium transporter that also confers methylammonium (ammonium analogue) toxicity, suggesting functional diversification. Surprisingly, both alleles require a combination of synonymous and nonsynonymous substitutions for their functionality. These results bring out a hitherto-unknown pathway of protein evolution and pave the way for not only understanding protein evolution but also interpreting single nucleotide polymorphisms (SNPs).
铵转运蛋白存在于生命的所有三个域中。它们经历了广泛的水平基因转移（HGT）、基因复制和功能多样化，因此为研究蛋白质进化提供了一个绝佳的范例。我们试图用酿酒酵母的直系同源物盘基网柄菌的来互补酿酒酵母的ΔΔΔ菌株（三缺失菌株），否则该菌株在浓度<3 mM的铵作为唯一氮源时无法生长。我们观察到在低铵培养基上生长时，不能互补酿酒酵母的三缺失菌株。我们从PCR产生的突变体质粒文库中分离出的两个突变衍生物（和），它们能够互补酿酒酵母的三缺失菌株。有三个非同义替换和两个同义替换，这对其功能是必需的。有两个非同义替换和一个同义替换，所有这些对功能都是必需的。有趣的是，AmtA M1转运铵但不赋予甲胺毒性，而AmtA M2转运铵并赋予甲胺毒性，这表明了功能多样化。初步的生化分析表明，这些突变体在构象以及铵转运机制上存在差异。这些有趣的结果清楚地表明，仅通过研究非同义替换和同义替换无法彻底了解蛋白质进化。上述观察结果对生物过程的各个方面都具有重要意义，并将进行详细讨论。基因复制后的功能多样化是蛋白质进化的主要驱动力之一。虽然非同义替换在蛋白质功能多样化中的作用已得到充分认识，但同义替换在蛋白质进化中的作用的知识仍处于起步阶段。通过功能互补，我们分离出了盘基网柄菌铵转运蛋白基因（）的两个功能等位基因，否则该基因在酿酒酵母中不能作为铵转运蛋白发挥作用。其中一个是铵转运蛋白，而另一个是铵转运蛋白，它也赋予甲铵（铵类似物）毒性，这表明了功能多样化。令人惊讶的是，这两个等位基因的功能都需要同义替换和非同义替换的组合。这些结果揭示了一条迄今为止未知的蛋白质进化途径，不仅为理解蛋白质进化铺平了道路，也为解释单核苷酸多态性（SNP）铺平了道路。
