title: Recombination-induced tag exchange (RITE) cassette series to monitor protein dynamics in Saccharomyces cerevisiae.
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, posttranslational modifications are lost and need to be reincorporated in the new molecules. Protein stability and mobility are also relevant for the 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 posttranslational 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.
authors.affiliation: Division of Gene Regulation, Netherlands Cancer Institute, Netherlands Proteomics Centre, 1066CX Amsterdam, The Netherlands.
authors.forename: Marit
authors.initials: M
authors.lastname: Terweij
authors.forename: Tibor
authors.initials: T
authors.lastname: van Welsem
authors.forename: Sjoerd
authors.initials: S
authors.lastname: van Deventer
authors.forename: Kitty F
authors.initials: KF
authors.lastname: Verzijlbergen
authors.forename: Victoria
authors.initials: V
authors.lastname: Menendez-Benito
authors.forename: David
authors.initials: D
authors.lastname: Ontoso
authors.forename: Pedro
authors.initials: P
authors.lastname: San-Segundo
authors.forename: Jacques
authors.initials: J
authors.lastname: Neefjes
authors.forename: Fred
authors.initials: F
authors.lastname: van Leeuwen
mesh_terms: D047369:Chromatin Immunoprecipitation; D055879:Gene Knock-In Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D019426:Integrases / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; C045073:Cre recombinase; D019426:Integrases
keywords: epitope tag; protein inheritance; protein turnover; pulse-chase