Recombination-induced tag exchange to track old and new proteins

Abstract

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.chromatin | histone | proteasome | protein dynamics | turnover P roteins are dynamic molecules. Their abundance is controlled by synthesis and degradation and they can be subject to posttranslational processing, modification, and demodification. In addition, most proteins are very mobile and undergo interactions with multiple other protein partners (1-4). However, little is known about the dynamics of proteins within macromolecular complexes in vivo (2, 4). Studying time-dependent changes in physical properties of proteins or protein turnover requires methods to distinguish resident (old) proteins from new proteins. Current methods that do so are usually based on fluorescent reporters or differential chemical labeling. For example, fluorescence recovery after photo bleaching relies on exchange of the old bleached protein by nonbleached proteins (1, 3, 4). Alternative methods involve time-dependent changes in fluorescence, nonspecific pulse-chase labeling of proteins with labeled amino acids, or labeling with chemical dyes that specifically bind to short tags (5-7). Although suitable for detection of proteins by microscopy or mass spectrometry, a limitation of these methods is that they do not provide a handle for biochemical analysis of old and new proteins and their complexes. To solve this problem and to eliminate the requirement for chemical labels or UV light we developed recombination-induced tag exchange (RITE), a method in which a genetic epitope tag is switched by transient induction of a site-specific recombinase. As a consequence, old and newly synthesized proteins are differentially tagged, which enables monitoring of protein dynamics by multiple techniques, as illustrated here. In contrast to inducible expression strategies (8-12), differential tagging by a time-controlled site-specific protease (13), or the labeling methods described above, RITE allows parallel detection and purification of old and new proteins under physiological conditions and over long periods of time.We used RITE to probe the stability of chromatin. Photobleaching experiments using histones tagged with fluorescent reporters suggest that chromatin is a static comp...

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

“…Several other methods capitalize on similar advantages such as other self-labeling or destructive enzymes (see Table 1). We would like to highlight one recently developed method named "Recombination Induced Tag Exchange" (RITE), which allows for similar applications as SNAP-tagging while using a fundamentally different strategy (Verzijlbergen et al, 2010). It uses recombination induced switching of expression of differentially tagged versions of the same gene.…”

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

“…The RITE technique is a highly versatile and reliable tool for studying protein replacement (Verzijlbergen et al 2010;MenendezBenito et al 2013;Terweij et al 2013). Here we adapted it to study histone turnover, but it can be used to study any protein with a relatively short mRNA half-life.…”

“…H3.2 RITE strain was constructed by PCR amplification of the HA/T7 RITE cassette (Verzijlbergen et al 2010) followed by integration at the endogenous locus by homologous recombination. The Cre-EBD open reading frame was PCR amplified from pTW040 (Verzijlbergen et al 2010) and recloned in pRAD15 vector (gift from Y. Watanabe) under an attenuated adh1 promoter. The plasmid was integrated at the ala1 locus at the MluI site.…”

“…The RITE method measures the actual protein turnover, as it detects proteins synthesized before and after a genetic epitope tag switching during the same experiment (Terweij et al 2013). This technique has been used in S. cerevisiae to track histone H3 during the cell cycle (Verzijlbergen et al 2010) and to track proteins that mark intracellular compartments (Menendez-Benito et al 2013).…”

Spatiotemporal analysis of organelle and macromolecular complex inheritance

“…Here we address these questions using a unique fluorescencebased system called "recombination-induced tag exchange" (RITE) (15,16). RITE is specifically designed to distinguish and simultaneously monitor endogenous expression of old and newly synthesized proteins.…”