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

Seok Ho Yu, Michael Boyce, Amberlyn M. Wands, Michelle R. Bond, Carolyn R. Bertozzi, Jennifer J. Kohler

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4834-4839
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number13
DOIs
StatePublished - Mar 27 2012

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Diazomethane
Nuclear Pore Complex Proteins
Cell Nucleus Active Transport
Proteins
Uridine Diphosphate
Acetylglucosamine
Post Translational Protein Processing
Ultraviolet Rays
Nuclear Proteins
Eukaryota
Mammals
Mass Spectrometry

Keywords

  • Diazirine
  • Glycosylation
  • Metabolism
  • Nucleoporins
  • Posttranslational modification

ASJC Scopus subject areas

  • General

Cite this

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners. / Yu, Seok Ho; Boyce, Michael; Wands, Amberlyn M.; Bond, Michelle R.; Bertozzi, Carolyn R.; Kohler, Jennifer J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, No. 13, 27.03.2012, p. 4834-4839.

Research output: Contribution to journalArticle

Yu, Seok Ho ; Boyce, Michael ; Wands, Amberlyn M. ; Bond, Michelle R. ; Bertozzi, Carolyn R. ; Kohler, Jennifer J. / Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners. In: Proceedings of the National Academy of Sciences of the United States of America. 2012 ; Vol. 109, No. 13. pp. 4834-4839.
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