Analyzing N-terminal arginylation through the use of peptide arrays and degradation assays

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dc.contributor.authorWadas, Brandon
dc.contributor.authorPiatkov, Konstantin I.
dc.contributor.authorBrower, Christopher S.
dc.contributor.authorVarshavsky, Alexander
dc.creator.orcidhttps://orcid.org/0000-0003-0289-5541
dc.date.accessioned2023-01-19T21:31:39Z
dc.date.available2023-01-19T21:31:39Z
dc.date.issued2016
dc.description.abstractNα-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.en_US
dc.identifier.citationThis is the published version of an article that is available at https://doi.org/10.1074/jbc.m116.747956. Recommended citation: Wadas, B., Piatkov, K. I., Brower, C. S., & Varshavsky, A. (2016). Analyzing N-terminal arginylation through the use of peptide arrays and degradation assays. Journal of Biological Chemistry, 291(40), 20976–20992. This item has been deposited in accordance with publisher copyright and licensing terms and with the author’s permission.en_US
dc.identifier.urihttps://hdl.handle.net/11274/14320
dc.identifier.urihttps://doi.org/10.1074/jbc.m116.747956
dc.language.isoen_USen_US
dc.publisherElsevieren_US
dc.rights.licenseCC-BY 4.0
dc.subjectProteasomeen_US
dc.subjectProtein degradationen_US
dc.subjectUbiquitinen_US
dc.subjectUbiquitin ligaseen_US
dc.subjectUbiquitylation (ubiquitination)en_US
dc.subjectAte1en_US
dc.subjectArginyltransferaseen_US
dc.subjectN-end ruleen_US
dc.titleAnalyzing N-terminal arginylation through the use of peptide arrays and degradation assaysen_US
dc.typeArticleen_US

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