In 1963, a group of researchers observed that specific radioactive amino acids were being incorporated into proteins obtained from ribosome-free cell and tissue extracts. This incorporation of amino acids into ribosome-lacking cells was first observed in prokaryotes using leucine (Leu) and phenylalanine (Phe), and was further discovered in mammalian liver extracts using arginine. The incorporation of other amino acids into ribosome-lacking cells failed to yield similar results, suggesting that the mechanism was specific to leucine and phenylalanine in bacteria and arginine in mammals. One of the most interesting aspects of arginylation is that the amino acids used for arginylation are transferred from aminoacyl tRNAs onto the target protein, without the use of any other translational components. This way of modifying proteins post-translationally does not occur in any other amino acid addition to proteins, such as in glycylation, glutamylation, and tyrosination making arginylation truly unique.     
Upon discovery of this modification and its mechanism, further research was performed to identify an enzyme or enzymes which promote this modification. After identifying the enzyme responsible for this modification in both plants and guinea-pig hair follicles, it was cloned and characterized in yeast and given the name ATE1 due to its ability. Later studies have also identified various genes which code for ATE1 enzymes in multiple species, leading to the conclusion that ATE1 is present in all eukaryotes.