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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.

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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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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

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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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