Tandem fluorescent protein timers for in vivo analysis of protein dynamics

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 seve...

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Bibliographic Details
Published in:Nature biotechnology 2012-07, Vol.30 (7), p.708-714
Main Authors: KHMELINSKII, Anton, KELLER, Philipp J, HUBER, Wolfgang, SCHIEBEL, Elmar, KNOP, Michael, BARTOSIK, Anna, MEURER, Matthias, BARRY, Joseph D, MARDIN, Balca R, KAUFMANN, Andreas, TRAUTMANN, Susanne, WACHSMUTH, Malte, PEREIRA, Gislene
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Diverse techniques
Fluorescence
Fluorescent proteins
Fundamental and applied biological sciences. Psychology
Green Fluorescent Proteins - chemistry
High-Throughput Screening Assays
Kinetics
Molecular and cellular biology
Nuclear Pore - metabolism
Nuclear Pore - ultrastructure
Protein Stability
Proteins
Proteins - metabolism
Proteolysis
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae - ultrastructure
Subcellular Fractions - metabolism
Subcellular Fractions - ultrastructure
Yeast
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Summary: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.
ISSN:1087-0156
1546-1696
DOI:10.1038/nbt.2281