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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    ABSTRACT: Specific degradation of proteins is essential for virtually all cellular processes and carried out predominantly by the proteasome. The proteasome is important for clearance of damaged cellular proteins. Damaged proteins accumulate over time and excess damaged proteins may aggregate and induce death of old cells. In yeast, the localization of the proteasome changes dramatically during aging, possibly in response to altered proteasome activity requirements. We followed two key parameters of this process: the distribution of proteasomes in nuclear and cytosolic compartments and the formation of cytoplasmic aggregate-like structures called proteasome storage granules (PSGs). While replicative young cells efficiently relocalized proteasomes from the nucleus to the cytoplasm and formed PSGs, replicative old cells are less efficient in relocalizing the proteasome and show less PSG. By a microscopy-based genome-wide screen, we identified genetic factors involved in these processes. Both relocalization of the proteasome and PSG formation were affected by two of the three N-acetylation complexes. These N-acetylation complexes also had different effects on the longevity of cells, indicating that each N-acetylation complex has different roles in proteasome location and aging.
    Journal of Cell Science 11/2014; 128(1). DOI:10.1242/jcs.157354 · 5.43 Impact Factor
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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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    ABSTRACT: Nuclear pore complexes (NPCs) are essential protein assemblies that span the nuclear envelope and establish nuclear-cytoplasmic compartmentalization. We have investigated mechanisms that control NPC number in mother and daughter cells during the asymmetric division of budding yeast. By simultaneously tracking existing NPCs and newly synthesized NPC protomers (nups) through anaphase, we uncovered a pool of the central channel nup Nsp1 that is actively targeted to the bud in association with endoplasmic reticulum. Bud targeting required an intact actin cytoskeleton and the class V myosin, Myo2. Selective inhibition of cytoplasmic Nsp1 or inactivation of Myo2 reduced the inheritance of NPCs in daughter cells, leading to a daughter-specific loss of viability. Our data are consistent with a model in which Nsp1 releases a barrier that otherwise prevents NPC passage through the bud neck. It further supports the finding that NPC inheritance, not de novo NPC assembly, is primarily responsible for controlling NPC number in daughter cells.
    The Journal of Cell Biology 10/2013; 203(2):215-32. DOI:10.1083/jcb.201305115 · 9.83 Impact Factor
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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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