Abstract
The sorting of soluble proteins for degradation in the vacuole is of vital importance in plant cells, and relies on the activity of vacuolar sorting receptors (VSRs). In the plant endomembrane system, VSRs bind vacuole-targeted proteins and facilitate their transport to the vacuole. Where exactly these interactions take place has remained controversial, however. Here, we examine the potential for VSR–ligand interactions in all compartments of the vacuolar transport system in tobacco mesophyll protoplasts. To do this, we developed compartment-specific VSR sensors that assemble as a result of a nanobody–epitope interaction, and monitored the degree of ligand binding by analysing Förster resonance energy transfer using fluorescence lifetime imaging microscopy (FRET–FLIM). We show that VSRs bind ligands in the endoplasmic reticulum (ER) and in the Golgi, but not in the trans-Golgi network/early endosome (TGN/EE) or multivesicular late endosomes, suggesting that the post-TGN/EE trafficking of ligands towards the vacuole is VSR independent. We verify this by showing that non-VSR–ligands are also delivered to the vacuole from the TGN/EE after endocytic uptake. We conclude that VSRs are required for the transport of ligands from the ER and the Golgi to the TGN/EE, and suggest that the onward transport to the vacuole occurs by default.
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Acknowledgements
We thank S. Peter (Institute of Physical and Theoretical Chemistry, University of Tübingen) for technical support and helpful discussions on FRET–FLIM. We would like to thank D. Vranjkovic and N. Gerling for technical help. The financial support of the Deutsche Forschungsgemeinschaft (PI 769/1-2 and the Collaborative Research Centre SFB 1101 ‘Molecular Encoding of Specificity in Plant Processes’ and TP A03) and the Deutscher Akademischer Austauschdienst (Project 57057314) is gratefully acknowledged.
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F.K., S.F., F.F. and P.P. designed and analysed the experiments. F.K., S.F., F.F. and B.L. performed the experiments. F.K. and P.P. wrote the manuscript.
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Künzl, F., Früholz, S., Fäßler, F. et al. Receptor-mediated sorting of soluble vacuolar proteins ends at the trans-Golgi network/early endosome. Nature Plants 2, 16017 (2016). https://doi.org/10.1038/nplants.2016.17
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DOI: https://doi.org/10.1038/nplants.2016.17
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