Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners.
Proc Natl Acad Sci U S A
; 109(13): 4834-9, 2012 Mar 27.
Article
em En
| MEDLINE
| ID: mdl-22411826
ABSTRACT
O-linked ß-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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Acetilglucosamina
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Coloração e Rotulagem
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Processamento de Proteína Pós-Traducional
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Reagentes de Ligações Cruzadas
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Complexo de Proteínas Formadoras de Poros Nucleares
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Luz
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article