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.

  • Letter
  • Published:

Chemical sensing of DNT by engineered olfactory yeast strain

Nature Chemical Biology volume 3, pages 325–330 (2007)Cite this article

Abstract

With the increasing threat of environmental toxicants including biological and chemical warfare agents, fabricating innovative biomimetic systems to detect these harmful agents is critically important. With the broad objective of developing such a biosensor, here we report the construction of a Saccharomyces cerevisiae strain containing the primary components of the mammalian olfactory signaling pathway. In this engineered yeast strain, WIF-1α, olfactory receptor signaling is coupled to green fluorescent protein expression. Using this 'olfactory yeast', we screened for olfactory receptors that could report the presence of the odorant 2,4-dinitrotoluene, an explosive residue mimic. With this approach, we have identified the novel rat olfactory receptor Olfr226, which is closely related to the mouse olfactory receptors Olfr2 and MOR226-1, as a 2,4-dinitrotoluene–responsive receptor.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Engineering of the WIF-1α strain.
Figure 2: GFP responses of WIF-1α–RI7 strain.
Figure 3: Shuttling of ligand-binding domains of other GPCRs in the WIF-1α system.
Figure 4: Screening of WIF-1α–OR strains for DNT-responsive colonies.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Buck, L. & Axel, R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65, 175–187 (1991).

    Article  CAS  Google Scholar 

  2. Mombaerts, P. Seven-transmembrane proteins as odorant and chemosensory receptors. Science 286, 707–711 (1999).

    Article  CAS  Google Scholar 

  3. Malnic, B., Hirono, J., Sato, T. & Buck, L.B. Combinatorial receptor codes for odors. Cell 96, 713–723 (1999).

    Article  CAS  Google Scholar 

  4. Jones, D.T. & Reed, R.R. Golf: an olfactory neuron specific G protein involved in odorant signal transduction. Science 244, 790–795 (1989).

    Article  CAS  Google Scholar 

  5. Bakalyar, H.A. & Reed, R.R. Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science 250, 1403–1406 (1990).

    Article  CAS  Google Scholar 

  6. Pace, U., Hanski, E., Salomon, Y. & Lancet, D. Odorant-sensitive adenylate cyclase may mediate olfactory reception. Nature 316, 255–258 (1985).

    Article  CAS  Google Scholar 

  7. Mori, K., Nagao, H. & Yoshihara, Y. The olfactory bulb: coding and processing of odor moleculae information. Science 286, 711–715 (1999).

    Article  CAS  Google Scholar 

  8. Firestein, S. How the olfactory system makes sense of scents. Nature 413, 211–218 (2001).

    Article  CAS  Google Scholar 

  9. Ladds, G., Goddard, A. & Davey, J. Functional analysis of heterologous GPCR signalling pathways in yeast. Trends Biotechnol. 23, 367–373 (2005).

    Article  CAS  Google Scholar 

  10. Pausch, M.H. G-protein coupled receptors in Saccharomyces cerevisiae: high throughput screening assays for drug discovery. Trends Biotechnol. 15, 487–494 (1997).

    Article  CAS  Google Scholar 

  11. Belluscio, L., Gold, G.H., Nemes, A. & Axel, R. Mice deficient in Golf are anosmic. Neuron 20, 69–81 (1998).

    Article  CAS  Google Scholar 

  12. Johnson, G.L. & Dhanasekaran, N. The G-Protein family and their interaction with receptors. Endocr. Rev. 10, 317–331 (1989).

    Article  CAS  Google Scholar 

  13. Crowe, M.L., Perry, B.N. & Connerton, I.F. Golf complements a GPA1 null mutation in Saccharomyces cerevisiae and functionally couples to the STE2 pheromone receptor. J. Recept. Signal Transduct. Res. 20, 61–73 (2000).

    Article  CAS  Google Scholar 

  14. Liang, J.J., Cockett, M. & Khawaja, X.Z. Immunohistochemical localization of G protein β1, β2, β3, β4, β5, and γ3 subunits in the adult rat brain. J. Neurochem. 71, 345–355 (1998).

    Article  CAS  Google Scholar 

  15. Asano, T. et al. Selective localization of G protein γ5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain. J. Neurochem. 79, 1129–1135 (2001).

    Article  CAS  Google Scholar 

  16. Russell, M., Bradshaw-Rouse, J., Markwardt, D. & Heideman, W. Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest. Mol. Biol. Cell 4, 757–765 (1993).

    Article  CAS  Google Scholar 

  17. Lalli, E. & Sassone-Corsi, P. Signal transduction and gene regulation: the nuclear response to cAMP. J. Biol. Chem. 269, 17359–17362 (1994).

    CAS  PubMed  Google Scholar 

  18. Price, L.A., Kajkowski, E.M., Hadcock, J.R., Ozenberger, B.A. & Pausch, M.H. Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway. Mol. Cell. Biol. 15, 6188–6195 (1995).

    Article  CAS  Google Scholar 

  19. King, K., Dohlman, H.G., Thorner, J., Caron, M.G. & Lefkowitz, R.J. Control of yeast mating signal transduction by a mammalian β2 adrenergic receptor and Gs α subunit. Science 250, 121–123 (1990).

    Article  CAS  Google Scholar 

  20. Miret, J.J., Rakhilina, L., Silverman, L. & Oehlen, B. Functional expression of heteromeric calcitonin gene-related peptide and adrenomedullin receptors in yeast. J. Biol. Chem. 277, 6881–6887 (2002).

    Article  CAS  Google Scholar 

  21. Krautwurst, D., Yau, K.W. & Reed, R.R. Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95, 917–926 (1998).

    Article  CAS  Google Scholar 

  22. Zhao, H. et al. Functional expression of a mammalian odorant receptor. Science 279, 237–242 (1998).

    Article  CAS  Google Scholar 

  23. Araneda, R.C., Peterlin, Z., Zhang, X., Chesler, A. & Firestein, S. A pharmacological profile of the aldehyde receptor repertoire in rat olfactory epithelium. J. Physiol. 555, 743–756 (2004).

    Article  CAS  Google Scholar 

  24. Shirokova, E. et al. Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants. J. Biol. Chem. 280, 11807–11815 (2005).

    Article  CAS  Google Scholar 

  25. Minic, J. et al. Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS J. 272, 524–537 (2005).

    Article  CAS  Google Scholar 

  26. Kobilka, B.K. et al. Chimeric α2-, β2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science 240, 1310–1316 (1988).

    Article  CAS  Google Scholar 

  27. Kajiya, K. et al. Molecular bases of odor discrimination: reconstitution of olfactory receptors that recognize overlapping sets of odorants. J. Neurosci. 21, 6018–6025 (2001).

    Article  CAS  Google Scholar 

  28. Hoffman, C.S. & Winston, F.A. Ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272 (1987).

    Article  CAS  Google Scholar 

  29. Wuestehube, L.J. & Schekman, R.W. Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast. Methods Enzymol. 219, 124–136 (1992).

    Article  CAS  Google Scholar 

  30. Radhika, V., Milkevitch, M., Audige, V., Proikas-Cezanne, T. & Dhanasekaran, N. Engineered Saccharomyces cerevisiae strain BioS-1, for the detection of water-borne toxic metal contaminants. Biotechnol. Bioeng. 90, 29–35 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank R. Reed (Johns Hopkins University School of Medicine), N. Gautam (Washington University School of Medicine) and J. Robishaw (Geisinger Clinic) for their kind gift of cDNA inserts encoding rat ACIII, β2 and γ5, respectively. Critical reading of the manuscript by J. Gardner, Z. Goldsmith and R. Saker is gratefully acknowledged. This work was sponsored by the US Defense Advanced Research Projects Agency through the Space and Naval Warfare Systems Center, San Diego, Contract No. N66001-00-C-8050.

Author information

Authors and Affiliations

  1. Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, 19140, Pennsylvania, USA

    Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran

Authors
  1. Venkat Radhika
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tassula Proikas-Cezanne
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Muralidharan Jayaraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Djamila Onesime
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ji Hee Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Danny N Dhanasekaran
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.R. carried out all the biochemical and fluorometric analyses. T.P.-C. made the receptor cassette and cloned Olfr226. M.J. cloned the receptors from rat olfactory epithelium. D.O. and J.H. assisted in the construction of the WIF-1 strain. D.D. conceived and supervised the project. All authors discussed the results, and D.D. wrote the manuscript.

Corresponding author

Correspondence to Danny N Dhanasekaran.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Amino acid sequences of rat Olfr226, mouse Olfr2 and mouse MOR226-1. (PDF 60 kb)

Supplementary Methods (PDF 101 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Radhika, V., Proikas-Cezanne, T., Jayaraman, M. et al. Chemical sensing of DNT by engineered olfactory yeast strain. Nat Chem Biol 3, 325–330 (2007). https://doi.org/10.1038/nchembio882

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio882

This article is cited by

Search

Advanced 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