Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Tandem fluorescent protein fusions as fluorescent timers.
Figure 2: Analysis of differential inheritance of cellular structures with the mCherry-sfGFP timer.
Figure 3: Inheritance of nuclear pore complexes during yeast mitosis.
Figure 4: Analysis of protein turnover with the mCherry-sfGFP tFT.
Figure 5: Identification of components of the N-end rule pathway.

References

  1. Schoenheimer, R. The Dynamic State of Body Constituents (Harvard University Press, Cambridge, MA, 1942).

  2. Nakayama, K.I. & Nakayama, K. Ubiquitin ligases: cell-cycle control and cancer. Nat. Rev. Cancer 6, 369–381 (2006).

    CAS  Article  Google Scholar 

  3. Johnston, J.A., Ward, C.L. & Kopito, R.R. Aggresomes: a cellular response to misfolded proteins. J. Cell Biol. 143, 1883–1898 (1998).

    CAS  Article  Google Scholar 

  4. Morimoto, R.I. Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. Genes Dev. 22, 1427–1438 (2008).

    CAS  Article  Google Scholar 

  5. Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    CAS  Article  Google Scholar 

  6. Pratt, J.M. et al. Dynamics of protein turnover, a missing dimension in proteomics. Mol. Cell. Proteomics 1, 579–591 (2002).

    CAS  Article  Google Scholar 

  7. Eden, E. et al. Proteome half-life dynamics in living human cells. Science 331, 764–768 (2011).

    CAS  Article  Google Scholar 

  8. Zhang, L. et al. Method for real-time monitoring of protein degradation at the single cell level. Biotechniques 42, 446–450 (2007).

    CAS  Article  Google Scholar 

  9. Yen, H.-C.S., Xu, Q., Chou, D.M., Zhao, Z. & Elledge, S.J. Global protein stability profiling in mammalian cells. Science 322, 918–923 (2008).

    CAS  Article  Google Scholar 

  10. Gordon, A. et al. Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nat. Methods 4, 175–181 (2007).

    CAS  Article  Google Scholar 

  11. Yen, H.-C.S. & Elledge, S.J. Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science 322, 923–929 (2008).

    CAS  Article  Google Scholar 

  12. Newman, R.H., Fosbrink, M.D. & Zhang, J. Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem. Rev. 111, 3614–3666 (2011).

    CAS  Article  Google Scholar 

  13. Wu, B., Piatkevich, K.D., Lionnet, T., Singer, R.H. & Verkhusha, V.V. Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics. Curr. Opin. Cell Biol. 23, 310–317 (2011).

    CAS  Article  Google Scholar 

  14. Terskikh, A. et al. “Fluorescent timer”: protein that changes color with time. Science 290, 1585–1588 (2000).

    CAS  Article  Google Scholar 

  15. Subach, F.V. et al. Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nat. Chem. Biol. 5, 118–126 (2009).

    CAS  Article  Google Scholar 

  16. Tsuboi, T., Kitaguchi, T., Karasawa, S., Fukuda, M. & Miyawaki, A. Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein. Mol. Biol. Cell 21, 87–94 (2010).

    CAS  Article  Google Scholar 

  17. Merzlyak, E.M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).

    CAS  Article  Google Scholar 

  18. Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T.C. & Waldo, G.S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    Article  Google Scholar 

  19. Pereira, G., Tanaka, T.U., Nasmyth, K. & Schiebel, E. Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex. EMBO J. 20, 6359–6370 (2001).

    CAS  Article  Google Scholar 

  20. Malínská, K., Malínský, J., Opekarová, M. & Tanner, W. Visualization of protein compartmentation within the plasma membrane of living yeast cells. Mol. Biol. Cell 14, 4427–4436 (2003).

    Article  Google Scholar 

  21. Takizawa, P.A., DeRisi, J.L., Wilhelm, J.E. & Vale, R.D. Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. Science 290, 341–344 (2000).

    CAS  Article  Google Scholar 

  22. Chen, T. et al. Multigenerational cortical inheritance of the Rax2 protein in orienting polarity and division in yeast. Science 290, 1975–1978 (2000).

    CAS  Article  Google Scholar 

  23. Strambio-De-Castillia, C., Niepel, M. & Rout, M.P. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat. Rev. Mol. Cell Biol. 11, 490–501 (2010).

    CAS  Article  Google Scholar 

  24. D'Angelo, M.A., Raices, M., Panowski, S.H. & Hetzer, M.W. Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell 136, 284–295 (2009).

    CAS  Article  Google Scholar 

  25. Zabel, U. et al. Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p. J. Cell Biol. 133, 1141–1152 (1996).

    CAS  Article  Google Scholar 

  26. Makio, T. et al. The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly. J. Cell Biol. 185, 459–473 (2009).

    CAS  Article  Google Scholar 

  27. Steinkraus, K.A., Kaeberlein, M. & Kennedy, B.K. Replicative aging in yeast: the means to the end. Annu. Rev. Cell Dev. Biol. 24, 29–54 (2008).

    CAS  Article  Google Scholar 

  28. Winey, M., Yarar, D., Giddings, T.H. & Mastronarde, D.N. Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes. Mol. Biol. Cell 8, 2119–2132 (1997).

    CAS  Article  Google Scholar 

  29. Khmelinskii, A., Keller, P.J., Lorenz, H., Schiebel, E. & Knop, M. Segregation of yeast nuclear pores. Nature 466, E1 (2010).

    CAS  Article  Google Scholar 

  30. Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).

    CAS  Article  Google Scholar 

  31. Verzijlbergen, K.F. et al. Recombination-induced tag exchange to track old and new proteins. Proc. Natl. Acad. Sci. USA 107, 64–68 (2010).

    CAS  Article  Google Scholar 

  32. Ouellet, J. & Barral, Y. Organelle segregation during mitosis: Lessons from asymmetrically dividing cells. J. Cell Biol. 196, 305–313 (2012).

    CAS  Article  Google Scholar 

  33. Varshavsky, A. The N-end rule pathway and regulation by proteolysis. Protein Sci. 20, 1298–1345 (2011).

    CAS  Article  Google Scholar 

  34. Belle, A., Tanay, A., Bitincka, L., Shamir, R. & O'Shea, E.K. Quantification of protein half-lives in the budding yeast proteome. Proc. Natl. Acad. Sci. USA 103, 13004–13009 (2006).

    CAS  Article  Google Scholar 

  35. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    CAS  Article  Google Scholar 

  36. Tong, A.H.Y. & Boone, C. High-throughput strain construction and systematic synthetic lethal screening in Saccharomyces cerevisiae. Methods Microbiol. 36, 369–386 (2007).

    CAS  Article  Google Scholar 

  37. Hwang, C.-S., Shemorry, A. & Varshavsky, A. N-terminal acetylation of cellular proteins creates specific degradation signals. Science 327, 973–977 (2010).

    CAS  Article  Google Scholar 

  38. Swanson, R., Locher, M. & Hochstrasser, M. A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation. Genes Dev. 15, 2660–2674 (2001).

    CAS  Article  Google Scholar 

  39. Leggett, D.S. et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell 10, 495–507 (2002).

    CAS  Article  Google Scholar 

  40. Hwang, C.-S., Shemorry, A., Auerbach, D. & Varshavsky, A. The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases. Nat. Cell Biol. 12, 1177–1185 (2010).

    CAS  Article  Google Scholar 

  41. Shaner, N.C., Patterson, G.H. & Davidson, M.W. Advances in fluorescent protein technology. J. Cell Sci. 120, 4247–4260 (2007).

    CAS  Article  Google Scholar 

  42. Hardy, S., Legagneux, V., Audic, Y. & Paillard, L. Reverse genetics in eukaryotes. Biol. Cell 102, 561–580 (2010).

    CAS  Article  Google Scholar 

  43. Skarnes, W.C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).

    CAS  Article  Google Scholar 

  44. Bogdanove, A.J. & Voytas, D.F. TAL effectors: customizable proteins for DNA targeting. Science 333, 1843–1846 (2011).

    CAS  Article  Google Scholar 

  45. Yewdell, J.W., Lacsina, J.R., Rechsteiner, M.C. & Nicchitta, C.V. Out with the old, in with the new? Comparing methods for measuring protein degradation. Cell Biol. Int. 35, 457–462 (2011).

    CAS  Article  Google Scholar 

  46. Alber, F. et al. The molecular architecture of the nuclear pore complex. Nature 450, 695–701 (2007).

    CAS  Article  Google Scholar 

  47. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004).

    CAS  Article  Google Scholar 

  48. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    CAS  Article  Google Scholar 

  49. Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    CAS  Article  Google Scholar 

  50. Bachmair, A., Finley, D. & Varshavsky, A. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179–186 (1986).

    CAS  Article  Google Scholar 

  51. Pau, G., Fuchs, F., Sklyar, O., Boutros, M. & Huber, W. EBImage–an R package for image processing with applications to cellular phenotypes. Bioinformatics 26, 979–981 (2010).

    CAS  Article  Google Scholar 

  52. Boutros, M., Brás, L.P. & Huber, W. Analysis of cell-based RNAi screens. Genome Biol. 7, R66 (2006).

    Article  Google Scholar 

  53. Malo, N., Hanley, J.A., Cerquozzi, S., Pelletier, J. & Nadon, R. Statistical practice in high-throughput screening data analysis. Nat. Biotechnol. 24, 167–175 (2006).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

M.K. conceived and, together with E.S., A.Kh. and P.J.K., designed the project. A.Kh., M.M., A.B., A.Ka., S.T. and P.J.K. did all yeast experiments. B.R.M. did mammalian cell work. G.P. contributed reagents. P.J.K., J.D.B., A.Kh., W.H., M.W. and M.K. developed theory. P.J.K., J.D.B. and A.Kh. developed analytical tools. M.K., A.Kh. and P.J.K. wrote the manuscript with input from E.S. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Elmar Schiebel or Michael Knop.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Notes 1–6, Supplementary Theory, Supplementary Tables 1,2 and Supplementary Figures 1–16 (PDF 2971 kb)

Supplementary Table 3

Results of the screens for components of the N-end rule pathway (XLS 866 kb)

Supplementary Movie 1

Movie corresponding to Figure 3d (i, ii) (MOV 556 kb)

Supplementary Movie 2

Movie corresponding to Figure 3d (iii) (MOV 1374 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2281

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing