Reconstitution of protein arginylation pathways in bacteria for robust identification and quantification.

ATE1 is a conserved enzyme that catalyzes the covalent addition of arginine to proteins bearing N-terminal or mid-chain Asp and Glu residues. N-terminal (Nt) arginylation can also occur on Cys, Asn, and Gln following enzymatic conversion, often marking proteins for degradation. Essential for development, this pathway contributes to protein quality control and stress responses. Despite growing insight into ATE1 structure and function, the mechanisms governing its substrate selectivity and coordination with upstream oxygenase and deamidase remain poorly defined. Here, we reconstitute the human processing cascades that generate Nt-arginylated proteins in E. coli, enabling step-resolved analysis of arginylation outcomes in a cellular context. By co-expressing human ADO, NTAN1, or NTAQ1 with ATE1 in a modular system, we achieved efficient conversion of Nt-Cys, Asn, and Gln into arginylation-permissive forms, recapitulating key features of upstream processing. Using this platform, we demonstrated that N-terminal processing is efficient and that ATE1 preferentially modifies protein N-termini over internal acidic residues. Mid-chain arginylation of α-synuclein was detectable but occurred at low frequency, with no major differences in site selectivity observed across the ATE1 isoforms tested. Together, this bacterial reconstitution system provides a scalable experimental platform for quantitative, protein-level analysis of ATE1 substrate specificity under defined conditions.