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

dc.contributor.author

Yu, Seok-Ho

dc.contributor.author

Boyce, Michael

dc.contributor.author

Wands, Amberlyn M

dc.contributor.author

Bond, Michelle R

dc.contributor.author

Bertozzi, Carolyn R

dc.contributor.author

Kohler, Jennifer J

dc.date.accessioned

2020-01-01T17:03:41Z

dc.date.available

2020-01-01T17:03:41Z

dc.date.issued

2012-03-12

dc.date.updated

2020-01-01T17:03:40Z

dc.description.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.

dc.identifier

1114356109

dc.identifier.issn

0027-8424

dc.identifier.issn

1091-6490

dc.identifier.uri

https://hdl.handle.net/10161/19697

dc.language

eng

dc.publisher

Proceedings of the National Academy of Sciences

dc.relation.ispartof

Proceedings of the National Academy of Sciences of the United States of America

dc.relation.isversionof

10.1073/pnas.1114356109

dc.subject

Hela Cells

dc.subject

Cell Nucleus

dc.subject

Humans

dc.subject

Diazomethane

dc.subject

Acetylglucosamine

dc.subject

Peptides

dc.subject

Nuclear Pore Complex Proteins

dc.subject

Uridine Diphosphate

dc.subject

Cross-Linking Reagents

dc.subject

Staining and Labeling

dc.subject

Protein Processing, Post-Translational

dc.subject

Mutagenesis

dc.subject

Repetitive Sequences, Amino Acid

dc.subject

Protein Binding

dc.subject

Active Transport, Cell Nucleus

dc.subject

Light

dc.subject

Models, Biological

dc.title

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

dc.type

Journal article

duke.contributor.orcid

Boyce, Michael|0000-0002-2729-4876

pubs.begin-page

4834

pubs.end-page

4839

pubs.issue

13

pubs.organisational-group

School of Medicine

pubs.organisational-group

Duke

pubs.organisational-group

Duke Cancer Institute

pubs.organisational-group

Institutes and Centers

pubs.organisational-group

Biochemistry

pubs.organisational-group

Basic Science Departments

pubs.publication-status

Published

pubs.volume

109

Files

Original bundle

Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Yu et al 2012.pdf
Size:
3.56 MB
Format:
Adobe Portable Document Format
Description:
Published version