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Peptides induce persistent signaling from endosomes by a nutrient transceptor

Nature Chemical Biology volume 8, pages 400–408 (2012)Cite this article

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Abstract

The yeast Gap1 transceptor mediates amino acid activation of the protein kinase A pathway and undergoes endocytic internalization following amino acid transport. We identified three specific γ-glutamyl dipeptides that cause persistent cyclic AMP–independent activation of protein kinase A, prevent Gap1 vacuolar sorting and cause Gap1 accumulation in endosomes. To our knowledge, these are the first examples of persistent agonists of a transceptor. In yeast mutants blocked in multivesicular body sorting, L-citrulline mimicked persistent signaling, further supporting that the internalized Gap1 transceptor keeps signaling. Unexpectedly, these dipeptides were transported by Gap1 and not by the regular dipeptide transporters. Their uptake was unusually sensitive to external pH and caused transient intracellular acidification. High external pH, NHA1 deletion or V-ATPase inhibition overcame the vacuolar sorting defect. Hence, this work has identified specific dipeptides that cause enhanced proton influx through the Gap1 symporter, resulting in its defective vacuolar sorting, and independently transform it into a persistently signaling transceptor.

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Figure 1: Major Gap1-dependent phenotypes associated with the addition of specific γ-glutamyl dipeptides to nitrogen-starved cells.
Figure 2: Persistent γ-glutamyl dipeptides induce Gap1-dependent intracellular acidification.
Figure 3: Strong pH dependence for activation of PKA targets, Gap1-mediated transport and defects in Gap1 vacuolar sorting by γ-glutamyl dipeptides.
Figure 4: Deletion of NHA1, which increased cytosolic pH, improved vacuolar sorting of Gap1 and abolished cytosol acidification and cAMP accumulation by γ-glutamyl dipeptides but not persistent PKA signaling.
Figure 5: Deletion of selected genes responsible for Gap1 MVB sorting mimics persistent PKA signaling with regular amino acids independently of Gap1 recycling.

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Acknowledgements

We would specially like to thank H. Sychrova and members of her laboratory, L. Maresova and O. Kinclova-Zimmermanova for their help and guidance in the intracellular pH measurements. We thank W. Verheyden and R. Wicik for technical help with the experiments, N. Vangoethem for help with preparation of the figures and V. Diaz for helping us with organochemistry calculations and concepts. We would also like to thank all researchers that have kindly provided us with strains, plasmids and antibodies from their laboratories (mentioned in Supplementary Tables 1 and 2). This work was supported by an European Commission–Marie Curie fellowship to M.R.-T., by Fonds Wetenschappelijk Onderzoek visiting and postdoctoral fellowships to M.R.-T. and G.V.Z. and by grants from the Fund for Scientific Research–Flanders, Interuniversity Attraction Poles Network P6/14, the Research Fund of the KU Leuven (Concerted Research Actions) and the Hercules Foundation (Flanders).

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Author notes

  1. Marta Rubio-Texeira and Griet Van Zeebroeck: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Flanders, Belgium

    Marta Rubio-Texeira, Griet Van Zeebroeck & Johan M Thevelein

  2. Department of Molecular Microbiology, VIB, Flanders, Belgium

    Marta Rubio-Texeira, Griet Van Zeebroeck & Johan M Thevelein

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  1. Marta Rubio-Texeira
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  2. Griet Van Zeebroeck
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Contributions

G.V.Z. and M.R.-T. performed the experimental work and contributed with J.M.T. to the design and discussion of the experimental work and the writing of the paper.

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Correspondence to Johan M Thevelein.

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The authors declare no competing financial interests.

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Rubio-Texeira, M., Van Zeebroeck, G. & Thevelein, J. Peptides induce persistent signaling from endosomes by a nutrient transceptor. Nat Chem Biol 8, 400–408 (2012). https://doi.org/10.1038/nchembio.910

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