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 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.
Verzijlbergen
Kitty F
KF
Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Menendez-Benito
Victoria
V
van Welsem
Tibor
T
van Deventer
Sjoerd J
SJ
Lindstrom
Derek L
DL
Ovaa
Huib
H
Neefjes
Jacques
J
Gottschling
Daniel E
DE
van Leeuwen
Fred
F
D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000235:genetics*; D005456:Fluorescent Dyes / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000502:physiology*; D015203:Reproducibility of Results
D002843:Chromatin; D000939:Epitopes; D005456:Fluorescent Dyes; D006657:Histones; D011506:Proteins; D011993:Recombinant Fusion Proteins; C045073:Cre recombinase; D019426:Integrases; D046988:Proteasome Endopeptidase Complex