title: 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 have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only α-amino groups of N-terminal residues but also γ-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 a substrate's canonical (Nt-arginylatable) N-terminal residue, 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 the 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.
authors: From the Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125.
authors: Brandon
authors: B
authors: Wadas
authors: the Center for Biotechnology and Biomedicine, Skolkovo Institute of Science and Technology, Moscow 143026, Russia, and.
authors: Konstantin I
authors: KI
authors: Piatkov
authors: the Department of Biology, Texas Woman's University, Denton, Texas 76204.
authors: Christopher S
authors: CS
authors: Brower
authors: From the Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, avarsh@caltech.edu.
authors: Alexander
authors: A
authors: Varshavsky
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D007527:Isoenzymes; D019881:Aminoacyltransferases; C019727:arginyltransferase
keywords: Ate1; N-end rule; arginyltransferase; proteasome; protein degradation; ubiquitin; ubiquitin ligase; ubiquitylation (ubiquitination)