Recombination-induced tag exchange to track old and new proteins

Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2009; 107(1):64-8. DOI: 10.1073/pnas.0911164107
Source: PubMed


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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Available from: Daniel Gottschling, Jun 09, 2014
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    • "Therefore, we tagged the b1 subunit (Pre3) of the proteasome with a fluorescent recombination induced tag exchange (RITE) cassette (Verzijlbergen et al., 2010), to differentially label new and old proteasomes. Integration of the RITE cassette behind the b1 gene results in a GFP-tagged proteasome produced before tag exchange, whereas new proteasomes [produced after tag exchange due to translocation of an estrogen receptor (ER)-coupled Cre-recombinase to the nucleus after addition of b-estradiol (Verzijlbergen et al., 2010)] will be labeled with mRFP. The genetic GFP-for-mRFP swapping is permanent and induced after two days of starvation. "
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    • "Leeuwen, Netherlands Cancer Institute, Amsterdam, Netherlands; Verzijlbergen et al., 2010) were sequentially replaced by mCherry and GFP genes, respectively . ORFs encoding NSP1 and NIC96 with their cognate promoters and 3 UTRs were inserted into pRS416 (Sikorski and Hieter, 1989) to generate PLPC19 and PLPC20. "
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    • "Background recombination before induction of the tag switch and recombination efficiency after induction can be determined by Southern blot analysis or by plating cells on nonselective media and then replica-plating the colonies to media containing Hygromycin (see Figure 3 and Verzijlbergen et al. 2010). In a typical experiment, the average background recombination is 10% or less, whereas the Cre-induced recombination efficiency is 95% or more (see Figure 3 and Verzijlbergen et al. 2010). The completion of a recombination-induced tag switch in a population of cells generally takes a few hours (Figure 3). "
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    ABSTRACT: Proteins are not static entities. They are highly mobile and their steady state levels are achieved by a balance between ongoing synthesis and degradation. The dynamic properties of a protein can have important consequences for its function. For example, when a protein is degraded and replaced by a newly synthesized one, post-translational modifications are lost and need to be reincorporated in the new molecules. Protein stability and mobility are also relevant for duplication of macromolecular structures or organelles, which involves coordination of protein inheritance with the synthesis and assembly of newly synthesized proteins. To measure protein dynamics we recently developed a genetic pulse-chase assay called Recombination-Induced Tag Exchange (RITE). RITE has been successfully used in Saccharomyces cerevisiae to measure turnover and inheritance of histone proteins, to study changes in post-translational modifications on aging proteins, and to visualize the spatiotemporal inheritance of protein complexes and organelles in dividing cells. Here we describe a series of successful RITE cassettes that are designed for biochemical analyses, genomics studies, as well as single cell fluorescence applications. Importantly, the genetic nature and the stability of the tag-switch offer the unique possibility to combine RITE with high-throughput screening for protein dynamics mutants and mechanisms. The RITE cassettes are widely applicable, modular by design, and can therefore be easily adapted for use in other cell types or organisms.
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