Recombination-induced tag exchange to track old and new proteins

The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequ...

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Vydáno v:Proceedings of the National Academy of Sciences - PNAS Ročník 107; číslo 1; s. 64
Hlavní autoři: Verzijlbergen, Kitty F, Menendez-Benito, Victoria, van Welsem, Tibor, van Deventer, Sjoerd J, Lindstrom, Derek L, Ovaa, Huib, Neefjes, Jacques, Gottschling, Daniel E, van Leeuwen, Fred
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 05.01.2010
Témata:
Chromatin - genetics
Chromatin - metabolism
Epitopes - genetics
Fluorescent Dyes - metabolism
Histones - genetics
Histones - metabolism
Integrases - genetics
Integrases - metabolism
Proteasome Endopeptidase Complex - metabolism
Proteins - genetics
Proteins - metabolism
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Recombination, Genetic - physiology
Reproducibility of Results
ISSN:1091-6490, 1091-6490
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Shrnutí:The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.
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ISSN:1091-6490
1091-6490
DOI:10.1073/pnas.0911164107