High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers

Tandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 2378; p. 85
Main Authors Fung, Jia Jun, Blöcher-Juárez, Karla, Khmelinskii, Anton
Format Journal Article
LanguageEnglish
Published United States 2022
Subjects
Kinetics
Proteolysis
Proteome - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Tandem fluorescent protein timers
Protein degradation
Proteostasis
tFT
Fluorescent proteins
Protein dynamics
Protein quality control
Protein turnover
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Summary:Tandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover of tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of protein turnover with tFTs in yeast using fluorescence measurements of ordered colony arrays. We describe guidelines on optimization of experimental design with regard to the layout of colony arrays, growth conditions, and instrument choice. Combined with semi-automated genetic crossing using synthetic genetic array (SGA) methodology and high-throughput protein tagging with SWAp-Tag (SWAT) libraries, this approach can be used to compare protein turnover across the proteome and to identify regulators of protein turnover genome-wide.
ISSN:1940-6029
DOI:10.1007/978-1-0716-1732-8_6