Tandem fluorescent protein timers for in vivo analysis of protein dynamics

Abstract

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

“…Also, protoplasts provide a shorter time frame for expression analyses (typically up to 48 h) and usually can be transformed only once. The longer expression time in infiltrated leaves (and the possibility of performing multiple infiltrations) allows avoidance of problems that might arise, for example, from different maturation times of fluorescent proteins (as known, for example, for GFP/YFP in comparison with mCherry; Khmelinskii et al, 2012).…”

Dynamic translation regulation in Caulobacter cell cycle control

“…7B). This construct enables identification of newly synthesized SpmX proteins by differentiating the sfGFP and the mCherry signals (32). As a control, we fused the C terminus of the constitutively expressed β′ subunit of RNA polymerase to the fluorescent timer.…”

Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis

“…In tFTs, the tandemly tagged fluorescent proteins that could switch colors during the time course of maturation were fused to individual model substrates. As different fluorophores need to be customized in tFTs to allow for monitoring the dynamics of proteins with distinct half-lives, its general applicability for high-throughput analysis is yet to be tested [15]. Therefore, novel tools need to be developed to overcome the limitations in the existing techniques.…”

“…Global Protein Stability assay (GPS) [11,14] and "tandem Fluorescent Timers" (tFTs) [15] are two of the most recent non-MS techniques for profiling protein stability. Both techniques quantitatively compare the fluorescence intensities of the co-expressed fluorescent proteins, with one fused to the protein of interest for test and the other for reference.…”