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.

  • Article
  • Published:

Transport and signaling via the amino acid binding site of the yeast Gap1 amino acid transceptor

Nature Chemical Biology volume 5, pages 45–52 (2009)Cite this article

Abstract

Transporter-related nutrient sensors, called transceptors, mediate nutrient activation of signaling pathways through the plasma membrane. The mechanism of action of transporting and nontransporting transceptors is unknown. We have screened 319 amino acid analogs to identify compounds that act on Gap1, a transporting amino acid transceptor in yeast that triggers activation of the protein kinase A pathway. We identified competitive and noncompetitive inhibitors of transport, either with or without agonist action for signaling, including nontransported agonists. Using substituted cysteine accessibility method (SCAM) analysis, we identified Ser388 and Val389 as being exposed into the amino acid binding site, and we show that agonist action for signaling uses the same binding site as used for transport. Our results provide the first insight, to our knowledge, into the mechanism of action of transceptors. They indicate that signaling requires a ligand-induced specific conformational change that may be part of but does not require the complete transport cycle.

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: Competitive inhibitors of Gap1 transport can have agonist action or no agonist action for signaling.
Figure 2: Noncompetitive inhibitors of Gap1 transport can have agonist action or no agonist action for signaling.
Figure 3: SCAM analysis in gap1 CAR domain mutants identifies amino acid residues important for transport and signaling and two residues exposed into the amino acid binding site of Gap1.
Figure 4: Gap1 uses the same amino acid binding site for transport and signaling.
Figure 5: Model of transceptor functioning involving a specific conformation for signaling.

Similar content being viewed by others

References

  1. Ozcan, S., Dover, J. & Johnston, M. Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. EMBO J. 17, 2566–2573 (1998).

    Article  CAS  Google Scholar 

  2. Didion, T., Regenberg, B., Jorgensen, M.U., Kielland-Brandt, M.C. & Andersen, H.A. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae. Mol. Microbiol. 27, 643–650 (1998).

    Article  CAS  Google Scholar 

  3. Iraqui, I. et al. Amino acid signaling in Saccharomyces cerevisiae: a permease-like sensor of external amino acids and F-Box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease. Mol. Cell. Biol. 19, 989–1001 (1999).

    Article  CAS  Google Scholar 

  4. Klasson, H., Fink, G.R. & Ljungdahl, P.O. Ssy1p and Ptr3p are plasma membrane components of a yeast system that senses extracellular amino acids. Mol. Cell. Biol. 19, 5405–5416 (1999).

    Article  CAS  Google Scholar 

  5. Holsbeeks, I., Lagatie, O., Van Nuland, A., Van de Velde, S. & Thevelein, J.M. The eukaryotic plasma membrane as a nutrient-sensing device. Trends Biochem. Sci. 29, 556–564 (2004).

    Article  CAS  Google Scholar 

  6. Biswas, K. & Morschhauser, J. The Mep2p ammonium permease controls nitrogen starvation-induced filamentous growth in Candida albicans. Mol. Microbiol. 56, 649–669 (2005).

    Article  CAS  Google Scholar 

  7. Lorenz, M.C. & Heitman, J. The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae. EMBO J. 17, 1236–1247 (1998).

    Article  CAS  Google Scholar 

  8. Hyde, R., Cwiklinski, E.L., MacAulay, K., Taylor, P.M. & Hundal, H.S. Distinct sensor pathways in the hierarchical control of SNAT2, a putative amino acid transceptor, by amino acid availability. J. Biol. Chem. 282, 19788–19798 (2007).

    Article  CAS  Google Scholar 

  9. Goberdhan, D.C., Meredith, D., Boyd, C.A. & Wilson, C. PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids. Development 132, 2365–2375 (2005).

    Article  CAS  Google Scholar 

  10. Hyde, R., Taylor, P.M. & Hundal, H.S. Amino acid transporters: roles in amino acid sensing and signalling in animal cells. Biochem. J. 373, 1–18 (2003).

    Article  CAS  Google Scholar 

  11. Lalonde, S. et al. The dual function of sugar carriers. Transport and sugar sensing. Plant Cell 11, 707–726 (1999).

    Article  CAS  Google Scholar 

  12. Thevelein, J.M. & de Winde, J.H. Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 33, 904–918 (1999).

    Article  CAS  Google Scholar 

  13. Thevelein, J.M. et al. Nutrient sensing systems for rapid activation of the protein kinase A pathway in yeast. Biochem. Soc. Trans. 33, 253–256 (2005).

    Article  CAS  Google Scholar 

  14. Donaton, M.C. et al. The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 50, 911–929 (2003).

    Article  CAS  Google Scholar 

  15. Van Nuland, A. et al. Ammonium permease-based sensing mechanism for rapid ammonium activation of the protein kinase A pathway in yeast. Mol. Microbiol. 59, 1485–1505 (2006).

    Article  CAS  Google Scholar 

  16. Giots, F., Donaton, M.C. & Thevelein, J.M. Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 47, 1163–1181 (2003).

    Article  CAS  Google Scholar 

  17. Grenson, M. Inactivation-reactivation process and repression of permease formation regulate several ammonia-sensitive permeases in the yeast Saccharomyces cerevisiae. Eur. J. Biochem. 133, 135–139 (1983).

    Article  CAS  Google Scholar 

  18. Hein, C. & Andre, B. A C-terminal di-leucine motif and nearby sequences are required for NH4(+)-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae. Mol. Microbiol. 24, 607–616 (1997).

    Article  CAS  Google Scholar 

  19. Stanbrough, M. & Magasanik, B. Transcriptional and posttranslational regulation of the general amino acid permease of Saccharomyces cerevisiae. J. Bacteriol. 177, 94–102 (1995).

    Article  CAS  Google Scholar 

  20. Andre, B. An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast 11, 1575–1611 (1995).

    Article  CAS  Google Scholar 

  21. Wipf, D. et al. Conservation of amino acid transporters in fungi, plants and animals. Trends Biochem. Sci. 27, 139–147 (2002).

    Article  CAS  Google Scholar 

  22. Gilstring, C.F. & Ljungdahl, P.O. A method for determining the in vivo topology of yeast polytopic membrane proteins demonstrates that Gap1p fully integrates into the membrane independently of Shr3p. J. Biol. Chem. 275, 31488–31495 (2000).

    Article  CAS  Google Scholar 

  23. Hu, L.A. & King, S.C. Functional sensitivity of polar surfaces on transmembrane helix 8 and cytoplasmic loop 8–9 of the Escherichia coli GABA (4-aminobutyrate) transporter encoded by gabP: mutagenic analysis of a consensus amphipathic region found in transporters from bacteria to mammals. Biochem. J. 330, 771–776 (1998).

    Article  CAS  Google Scholar 

  24. King, S.C., Hu, L.A. & Pugh, A. Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP. Biochem. J. 376, 645–653 (2003).

    Article  CAS  Google Scholar 

  25. Wu, B. et al. Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p. J. Cell Biol. 173, 327–331 (2006).

    Article  CAS  Google Scholar 

  26. App, H. & Holzer, H. Purification and characterization of neutral trehalase from the yeast ABYS1 mutant. J. Biol. Chem. 264, 17583–17588 (1989).

    CAS  PubMed  Google Scholar 

  27. Durnez, P. et al. Activation of trehalase during growth induction by nitrogen sources in the yeast Saccharomyces cerevisiae depends on the free catalytic subunits of cAMP-dependent protein kinase, but not on functional Ras proteins. Yeast 10, 1049–1064 (1994).

    Article  CAS  Google Scholar 

  28. Hirimburegama, K. et al. Nutrient-induced activation of trehalase in nutrient-starved cells of the yeast Saccharomyces cerevisiae: cAMP is not involved as second messenger. J. Gen. Microbiol. 138, 2035–2043 (1992).

    Article  CAS  Google Scholar 

  29. Uno, I., Matsumoto, K., Adachi, K. & Ishikawa, T. Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast. J. Biol. Chem. 258, 10867–10872 (1983).

    CAS  PubMed  Google Scholar 

  30. Ma, P., Wera, S., Van Dijck, P. & Thevelein, J.M. The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol. Biol. Cell 10, 91–104 (1999).

    Article  CAS  Google Scholar 

  31. Mbonyi, K., van Aelst, L., Arguelles, J.C., Jans, A.W. & Thevelein, J.M. Glucose-induced hyperaccumulation of cyclic AMP and defective glucose repression in yeast strains with reduced activity of cyclic AMP-dependent protein kinase. Mol. Cell. Biol. 10, 4518–4523 (1990).

    Article  CAS  Google Scholar 

  32. De Craene, J.O., Soetens, O. & Andre, B. The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease. J. Biol. Chem. 276, 43939–43948 (2001).

    Article  CAS  Google Scholar 

  33. Risinger, A.L., Cain, N.E., Chen, E.J. & Kaiser, C.A. Activity-dependent reversible inactivation of the general amino acid permease. Mol. Biol. Cell 17, 4411–4419 (2006).

    Article  CAS  Google Scholar 

  34. Rubio-Texeira, M. & Kaiser, C.A. Amino acids regulate retrieval of the yeast general amino acid permease from the vacuolar targeting pathway. Mol. Biol. Cell 17, 3031–3050 (2006).

    Article  CAS  Google Scholar 

  35. Springael, J.Y. & Andre, B. Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae. Mol. Biol. Cell 9, 1253–1263 (1998).

    Article  CAS  Google Scholar 

  36. Grenson, M., Hou, C. & Crabeel, M. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J. Bacteriol. 103, 770–777 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Poulsen, P., Gaber, R.F. & Kielland-Brandt, M.C. Hyper- and hyporesponsive mutant forms of the Saccharomyces cerevisiae Ssy1 amino acid sensor. Mol. Membr. Biol. 25, 164–176 (2008).

    Article  CAS  Google Scholar 

  38. Ozcan, S., Dover, J., Rosenwald, A.G., Wolfl, S. & Johnston, M. Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression. Proc. Natl. Acad. Sci. USA 93, 12428–12432 (1996).

    Article  CAS  Google Scholar 

  39. Schwoppe, C., Winkler, H.H. & Neuhaus, H.E. Properties of the glucose-6-phosphate transporter from Chlamydia pneumoniae (HPTcp) and the glucose-6-phosphate sensor from Escherichia coli (UhpC). J. Bacteriol. 184, 2108–2115 (2002).

    Article  CAS  Google Scholar 

  40. Schwoppe, C., Winkler, H.H. & Neuhaus, H.E. Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli. Eur. J. Biochem. 270, 1450–1457 (2003).

    Article  CAS  Google Scholar 

  41. Faham, S. et al. The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321, 810–814 (2008).

    Article  CAS  Google Scholar 

  42. Holyoake, J. & Sansom, M.S. Conformational change in an MFS protein: MD simulations of LacY. Structure 15, 873–884 (2007).

    Article  CAS  Google Scholar 

  43. Yamashita, A., Singh, S.K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl–dependent neurotransmitter transporters. Nature 437, 215–223 (2005).

    Article  CAS  Google Scholar 

  44. Bechet, J., Greenson, M. & Wiame, J.M. Mutations affecting the repressibility of arginine biosynthetic enzymes in Saccharomyces cerevisiae. Eur. J. Biochem. 12, 31–39 (1970).

    Article  CAS  Google Scholar 

  45. Jauniaux, J.C. & Grenson, M. GAP1, the general amino acid permease gene of Saccharomyces cerevisiae. Nucleotide sequence, protein similarity with the other bakers yeast amino acid permeases, and nitrogen catabolite repression. Eur. J. Biochem. 190, 39–44 (1990).

    Article  CAS  Google Scholar 

  46. Bonneaud, N. et al. A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7, 609–615 (1991).

    Article  CAS  Google Scholar 

  47. Tamas, M.J., Rep, M., Thevelein, J.M. & Hohmann, S. Stimulation of the yeast high osmolarity glycerol (HOG) pathway: evidence for a signal generated by a change in turgor rather than by water stress. FEBS Lett. 472, 159–165 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank W. Verheyden and R. Wicik for technical help with the experiments, N. Vangoethem for help with preparation of the figures and B. André (Université Libre de Bruxelles) for the kind gift of strains, plasmids and antibodies. This work was supported by a Return Grant from the Belgian Federal Science Policy Office to M.V. and by grants from the Fund for Scientific Research - Flanders, Interuniversity Attraction Poles Network P5/30 and P6/14 and the Research Fund of the Katholieke Universiteit Leuven (Concerted Research Actions).

Author information

Authors and Affiliations

  1. Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium

    Griet Van Zeebroeck, Beatriz Monge Bonini, Matthias Versele & Johan M Thevelein

  2. Department of Molecular Microbiology, Vlaams Instituut voor Biotechnologie, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium

    Griet Van Zeebroeck, Beatriz Monge Bonini, Matthias Versele & Johan M Thevelein

Authors
  1. Griet Van Zeebroeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Beatriz Monge Bonini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias Versele
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johan M Thevelein
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.V.Z. and B.M.B. contributed mainly to the execution of the experimental work; M.V. and J.M.T. contributed to the design and discussion of the experimental work.

Corresponding author

Correspondence to Johan M Thevelein.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Discussion (PDF 475 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Van Zeebroeck, G., Bonini, B., Versele, M. et al. Transport and signaling via the amino acid binding site of the yeast Gap1 amino acid transceptor. Nat Chem Biol 5, 45–52 (2009). https://doi.org/10.1038/nchembio.132

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.132

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