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A cytosolic trans-activation domain essential for ammonium uptake

Abstract

Polytopic membrane proteins are essential for cellular uptake and release of nutrients. To prevent toxic accumulation, rapid shut-off mechanisms are required. Here we show that the soluble cytosolic carboxy terminus of an oligomeric ammonium transporter from Arabidopsis thaliana serves as an allosteric regulator essential for function; mutations in the C-terminal domain, conserved between bacteria, fungi and plants, led to loss of transport activity. When co-expressed with intact transporters, mutants inactivated functional subunits, but left their stability unaffected. Co-expression of two inactive transporters, one with a defective pore, the other with an ablated C terminus, reconstituted activity. The crystal structure of an Archaeoglobus fulgidus ammonium transporter (AMT)1 suggests that the C terminus interacts physically with cytosolic loops of the neighbouring subunit. Phosphorylation of conserved sites in the C terminus2 are proposed as the cognate control mechanism. Conformational coupling between monomers provides a mechanism for tight regulation, for increasing the dynamic range of sensing and memorizing prior events, and may be a general mechanism for transporter regulation.

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Figure 1: Arabidopsis AMT1;1 mutant functionality measured by their ability to confer growth to DL1 (Δ gap1 Δ mep1-3 ) on 2 mM ammonium as sole N-source.
Figure 2: Functional characterization of AMT1;1 mutants.
Figure 3: Structural model of Arabidopsis AMT1;1.
Figure 4: Co-expression of the trans- activation-deficient T460A and ammonium-recruitment-deficient D198N mutants reconstitutes a functional complex.

References

  1. Andrade, S. L., Dickmanns, A., Ficner, R. & Einsle, O. Crystal structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus. Proc. Natl Acad. Sci. USA 102, 14994–14999 (2005)

    ADS  CAS  Article  Google Scholar 

  2. Nühse, T. S., Stensballe, A., Jensen, O. N. & Peck, S. C. Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16, 2394–2405 (2004)

    Article  Google Scholar 

  3. Blakey, D. et al. Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry. Biochem. J. 364, 527–535 (2002)

    CAS  Article  Google Scholar 

  4. Ludewig, U. et al. Homo- and hetero-oligomerization of ammonium transporter-1 NH4+ uniporters. J. Biol. Chem. 278, 45603–45610 (2003)

    CAS  Article  Google Scholar 

  5. Khademi, S. et al. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science 305, 1587–1594 (2004)

    ADS  CAS  Article  Google Scholar 

  6. Veenhoff, L. M., Heuberger, E. H. & Poolman, B. Quaternary structure and function of transport proteins. Trends Biochem. Sci. 27, 242–249 (2002)

    CAS  Article  Google Scholar 

  7. Jiang, Y. et al. X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41 (2003)

    ADS  CAS  Article  Google Scholar 

  8. Borgnia, M., Nielsen, S., Engel, A. & Agre, P. Cellular and molecular biology of the aquaporin water channels. Annu. Rev. Biochem. 68, 425–458 (1999)

    CAS  Article  Google Scholar 

  9. Grasberger, B., Minton, A. P., DeLisi, C. & Metzger, H. Interaction between proteins localized in membranes. Proc. Natl Acad. Sci. USA 83, 6258–6262 (1986)

    ADS  CAS  Article  Google Scholar 

  10. Hamill, S., Cloherty, E. K. & Carruthers, A. The human erythrocyte sugar transporter presents two sugar import sites. Biochemistry 38, 16974–16983 (1999)

    CAS  Article  Google Scholar 

  11. Marini, A. M., Springael, J. Y., Frommer, W. B. & André, B. Cross-talk between ammonium transporters in yeast and interference by the soybean SAT1 protein. Mol. Microbiol. 35, 378–385 (2000)

    CAS  Article  Google Scholar 

  12. Schulman, H., Hanson, P. I. & Meyer, T. Decoding calcium signals by multifunctional CaM kinase. Cell Calcium 13, 401–411 (1992)

    CAS  Article  Google Scholar 

  13. Coutts, G., Thomas, G., Blakey, D. & Merrick, M. Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB. EMBO J. 21, 536–545 (2002)

    CAS  Article  Google Scholar 

  14. Conroy, M. J. et al. Electron and atomic force microscopy of the trimeric ammonium transporter AmtB. EMBO Rep. 5, 1153–1158 (2004)

    CAS  Article  Google Scholar 

  15. Zen, K. H., McKenna, E., Bibi, E., Hardy, D. & Kaback, H. R. Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains. Biochemistry 33, 8198–8206 (1994)

    CAS  Article  Google Scholar 

  16. Reinders, A. et al. Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays. Structure 10, 763–772 (2002)

    CAS  Article  Google Scholar 

  17. Schulze, W. X., Reinders, A., Ward, J., Lalonde, S. & Frommer, W. B. Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system. BMC Biochem. 4, 3 (2003)

    Article  Google Scholar 

  18. Kronzucker, H. J., Siddiqi, M. Y. & Glass, A. Kinetics of NH4+ influx in spruce. Plant Physiol. 110, 773–779 (1996)

    CAS  Article  Google Scholar 

  19. Rawat, S. R., Silim, S. N., Kronzucker, H. J., Siddiqi, M. Y. & Glass, A. D. AtAMT1 gene expression and NH4+ uptake in roots of Arabidopsis thaliana: evidence for regulation by root glutamine levels. Plant J. 19, 143–152 (1999)

    CAS  Article  Google Scholar 

  20. Marini, A. M., Soussi-Boudekou, S., Vissers, S. & Andre, B. A family of ammonium transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 4282–4293 (1997)

    CAS  Article  Google Scholar 

  21. Ludewig, U., von Wiren, N. & Frommer, W. B. Uniport of NH4+ by the root hair plasma membrane ammonium transporter LeAMT1;1. J. Biol. Chem. 277, 13548–13555 (2002)

    CAS  Article  Google Scholar 

  22. Ninnemann, O., Jauniaux, J. C. & Frommer, W. B. Identification of a high affinity NH4+ transporter from plants. EMBO J. 13, 3464–3471 (1994)

    CAS  Article  Google Scholar 

  23. Javelle, A., Severi, E., Thornton, J. & Merrick, M. Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB–GlnK complex formation. J. Biol. Chem. 279, 8530–8538 (2004)

    CAS  Article  Google Scholar 

  24. Marini, A. M., Boeckstaens, M., Benjelloun, F., Cherif-Zahar, B. & Andre, B. Structural involvement in substrate recognition of an essential aspartate residue conserved in Mep/Amt and Rh-type ammonium transporters. Curr. Genet. 49, 364–374 (2006)

    CAS  Article  Google Scholar 

  25. Changeux, J. P. & Edelstein, S. J. Allosteric mechanisms of signal transduction. Science 308, 1424–1428 (2005)

    ADS  CAS  Article  Google Scholar 

  26. Törnroth-Horsefield, S. et al. Structural mechanism of plant aquaporin gating. Nature 439, 688–694 (2006)

    ADS  Article  Google Scholar 

  27. Palmgren, M. G., Sommarin, M., Serrano, R. & Larsson, C. Identification of an autoinhibitory domain in the C-terminal region of the plant plasma membrane H+-ATPase. J. Biol. Chem. 266, 20470–20475 (1991)

    CAS  PubMed  Google Scholar 

  28. Curran, A. C. et al. Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain. J. Biol. Chem. 275, 30301–30308 (2000)

    CAS  Article  Google Scholar 

  29. Pittman, J. K. & Hirschi, K. D. Regulation of CAX1, an Arabidopsis Ca2+/H+ antiporter. Identification of an N-terminal autoinhibitory domain. Plant Physiol. 127, 1020–1029 (2001)

    CAS  Article  Google Scholar 

  30. Levy, J. et al. A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses. Science 303, 1361–1364 (2004)

    ADS  CAS  Article  Google Scholar 

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Acknowledgements

We would like to thank L. Yuan (University of Hohenheim) for the Arabidopsis AMT1;1 antiserum. This work was made possible by grants from the Department of Energy and the European Science award from the Körber Foundation to W.B.F.

Author Contributions D.L. created all mutants, developed the co-expression system and did the protein gel blots, S.L. generated GFP fusions and did the imaging, L.L.L. performed structural modelling, N.vW. contributed to production of the serum and was involved in developing the concept. All authors contributed sections of the manuscript. W.B.F. is responsible for the experimental design, developed the hypotheses, and interpreted the results.

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Correspondence to W. B. Frommer.

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This file contains Supplementary Methods, Supplementary Table S1, Supplementary Figures S1-S16 with Legends and additional references. (PDF 1843 kb)

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Loqué, D., Lalonde, S., Looger, L. et al. A cytosolic trans-activation domain essential for ammonium uptake. Nature 446, 195–198 (2007). https://doi.org/10.1038/nature05579

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