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.
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We are grateful to Y. Belyaev, S. Terjung and the Advanced Light Microscopy Facility of EMBL for support with microscopy; A. Riddell, A.P. Gonzalez and the FACS Facility of EMBL for flow cytometry analyses; the CellNetworks cluster for funding to M.K., the German Research Foundation for funding to E.S. (SFB638), the Howard Hughes Medical Institute for funding to P.J.K., the European Molecular Biology Organization for funding to A.Kh. (EMBO ALTF 1124-2010), the EU-FP7 Network of Excellence in Systems Microscopy for funding to J.D.B. and W.H., and the Novartis Stiftung for funding to A.Ka. We thank P.I. Bastiaens, D. Gilmour and A. Kinkhabwala for discussions.
The authors declare no competing financial interests.
Supplementary Methods, Supplementary Notes 1–6, Supplementary Theory, Supplementary Tables 1,2 and Supplementary Figures 1–16 (PDF 2971 kb)
Results of the screens for components of the N-end rule pathway (XLS 866 kb)
Movie corresponding to Figure 3d (i, ii) (MOV 556 kb)
Movie corresponding to Figure 3d (iii) (MOV 1374 kb)
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Khmelinskii, A., Keller, P., Bartosik, A. et al. Tandem fluorescent protein timers for in vivo analysis of protein dynamics. Nat Biotechnol 30, 708–714 (2012). https://doi.org/10.1038/nbt.2281
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