Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners.

Published

Journal Article

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

Full Text

Duke Authors

Cited Authors

  • Yu, S-H; Boyce, M; Wands, AM; Bond, MR; Bertozzi, CR; Kohler, JJ

Published Date

  • March 27, 2012

Published In

Volume / Issue

  • 109 / 13

Start / End Page

  • 4834 - 4839

PubMed ID

  • 22411826

Pubmed Central ID

  • 22411826

Electronic International Standard Serial Number (EISSN)

  • 1091-6490

Digital Object Identifier (DOI)

  • 10.1073/pnas.1114356109

Language

  • eng

Conference Location

  • United States