Analyzing N-terminal Arginylation Through the Use of Peptide Arrays and Degradation Assays

Abstract

Nα-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified (canonical) residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu and (oxidized) Cys. Over the last decade, several publications suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only alpha-amino groups of N-terminal residues but also gamma-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of canonical (Nt-arginylatable) N-terminal residue of a substrate, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about specificity of some Ate1 isoforms for N terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.

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