Abstract
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.
Highlights
Along the journey that starts from a nascent polypeptide, proteins undertake a series of intricate but error-prone steps including folding, trafficking, and assembly into complexes
There is a global decline of proteostasis and accumulation of abnormal proteins during aging [1, 2, 4], highlighting the importance of understanding how abnormal proteins are recognized and defining the machinery involved in their selective degradation
We describe a protocol for high-throughput analysis of protein turnover with Tandem fluorescent protein timers (tFTs) using fluorescence measurements of ordered colony arrays grown on solid agar medium
Summary
Along the journey that starts from a nascent polypeptide, proteins undertake a series of intricate but error-prone steps including folding, trafficking, and assembly into complexes. We detail how to assemble array plates with layouts suitable for different applications, how to perform fluorescence measurements with a scanning plate reader, and how to correct and normalize the data These procedures can be integrated with high-throughput protein tagging using SWAT [27, 30, 32] or applied to the first genome-wide tFT library, constructed with the mCherry-sfGFP timer [19], to analyze the turnover of the yeast proteome in different environmental conditions. They can be combined with semi-automated genetic crossing using synthetic genetic array (SGA) methodology [33, 34] to screen for factors involved in the turnover of a protein of interest by examining the impact of different genetic perturbation on its behavior [5, 19]
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