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Receptor-mediated sorting of soluble vacuolar proteins ends at the trans-Golgi network/early endosome

Nature Plants volume 2, Article number: 16017 (2016) Cite this article

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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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Figure 1: Compartment-specific targeting of luminal ligand-binding domains (LBDs) in the plant endomembrane system through nanobody–epitope interactions.
Figure 2: All assembled VSR sensors are competent for ligand binding.
Figure 3: Analysis of VSR–ligand interaction identifies the ER as a compartment that favours ligand binding whereas the MVB/LE restricts ligand binding.
Figure 4: The Golgi provides ligand-binding conditions for VSRs.
Figure 5: The TGN/EE does not provide ligand-binding conditions for VSRs.
Figure 6: Vacuolar delivery of endocytosed soluble proteins does not depend on sorting signals.

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References

  1. Holwerda, B. C., Padgett, H. S. & Rogers, J. C. Proaleurain vacuolar targeting is mediated by short contiguous peptide interactions. Plant Cell 4, 307–318 (1992).

    Article  CAS  Google Scholar 

  2. Kirsch, T., Paris, N., Butler, J. M., Beevers, L. & Rogers, J. C. Purification and initial characterization of a potential plant vacuolar targeting receptor. Proc. Natl Acad. Sci. USA 91, 3403–3407 (1994).

    Article  CAS  Google Scholar 

  3. Robinson, D. G. & Pimpl, P. Receptor-mediated transport of vacuolar proteins: a critical analysis and a new model. Protoplasma 251, 247–264 (2014).

    Article  CAS  Google Scholar 

  4. Paris, N. et al. Molecular cloning and further characterization of a probable plant vacuolar sorting receptor. Plant Physiol. 115, 29–39 (1997).

    Article  CAS  Google Scholar 

  5. Ahmed, S. U., Bar-Peled, M. & Raikhel, N. V. Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells. Plant Physiol. 114, 325–336 (1997).

    Article  CAS  Google Scholar 

  6. De Marcos Lousa, C., Gershlick, D. C. & Denecke, J. Mechanisms and concepts paving the way towards a complete transport cycle of plant vacuolar sorting receptors. Plant Cell 24, 1714–1732 (2012).

    Article  CAS  Google Scholar 

  7. Cao, X., Rogers, S. W., Butler, J., Beevers, L. & Rogers, J. C. Structural requirements for ligand binding by a probable plant vacuolar sorting receptor. Plant Cell 12, 493–506 (2000).

    Article  CAS  Google Scholar 

  8. Luo, F. et al. How vacuolar sorting receptor proteins interact with their cargo proteins: crystal structures of apo and cargo-bound forms of the protease-associated domain from an Arabidopsis vacuolar sorting receptor. Plant Cell 26, 3693–3708 (2014).

    Article  CAS  Google Scholar 

  9. DaSilva, L. L., Foresti, O. & Denecke, J. Targeting of the plant vacuolar sorting receptor BP80 is dependent on multiple sorting signals in the cytosolic tail. Plant Cell 18, 1477–1497 (2006).

    Article  CAS  Google Scholar 

  10. Kim, H. et al. Homomeric interaction of AtVSR1 is essential for its function as a vacuolar sorting receptor. Plant Physiol. 154, 134–148 (2010).

    Article  CAS  Google Scholar 

  11. Saint-Jean, B., Seveno-Carpentier, E., Alcon, C., Neuhaus, J. M. & Paris, N. The cytosolic tail dipeptide Ile-Met of the pea receptor BP80 is required for recycling from the prevacuole and for endocytosis. Plant Cell 22, 2825–2837 (2010).

    Article  CAS  Google Scholar 

  12. Dettmer, J., Hong-Hermesdorf, A., Stierhof, Y. D. & Schumacher, K. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18, 715–730 (2006).

    Article  CAS  Google Scholar 

  13. Lam, S. K. et al. BFA-induced compartments from the Golgi apparatus and trans-Golgi network/early endosome are distinct in plant cells. Plant J. 60, 865–881 (2009).

    Article  CAS  Google Scholar 

  14. Foresti, O. & Denecke, J. Intermediate organelles of the plant secretory pathway: identity and function. Traffic 9, 1599–1612 (2008).

    Article  CAS  Google Scholar 

  15. Shen, J. et al. Organelle pH in the Arabidopsis endomembrane system. Mol. Plant 6, 1419–1437 (2013).

    Article  CAS  Google Scholar 

  16. Martiniere, A. et al. In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system. Plant Cell 25, 4028–4043 (2013).

    Article  CAS  Google Scholar 

  17. Luo, Y. et al. V-ATPase activity in the TGN/EE is required for exocytosis and recycling in Arabidopsis. Nature Plants 1, 15094 (2015).

    Article  CAS  Google Scholar 

  18. Niemes, S. et al. Retromer recycles vacuolar sorting receptors from the trans-Golgi network. Plant J. 61, 107–121 (2010).

    Article  CAS  Google Scholar 

  19. Stierhof, Y. D., Viotti, C., Scheuring, D., Sturm, S. & Robinson, D. G. Sorting nexins 1 and 2a locate mainly to the TGN. Protoplasma 250, 235–240 (2013).

    Article  CAS  Google Scholar 

  20. Scheuring, D. et al. Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23, 3463–3481 (2011).

    Article  CAS  Google Scholar 

  21. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).

    Article  CAS  Google Scholar 

  22. Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nature Methods 3, 887–889 (2006).

    Article  CAS  Google Scholar 

  23. Kubala, M. H., Kovtun, O., Alexandrov, K. & Collins, B. M. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci. 19, 2389–2401 (2010).

    Article  CAS  Google Scholar 

  24. Bucherl, C. A., Bader, A., Westphal, A. H., Laptenok, S. P. & Borst, J. W. FRET–FLIM applications in plant systems. Protoplasma 251, 383–394 (2014).

    Article  Google Scholar 

  25. Kriechbaumer, V. et al. Reticulomics: Protein-protein interaction studies with two plasmodesmata-localised reticulon family proteins identify binding partners enriched at plasmodesmata, ER and the plasma membrane. Plant Physiol. 169, 1933–1945 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Martiniere, A., Desbrosses, G., Sentenac, H. & Paris, N. Development and properties of genetically encoded pH sensors in plants. Front. Plant Sci. 4, 523 (2013).

    PubMed  PubMed Central  Google Scholar 

  27. Niemes, S. et al. Sorting of plant vacuolar proteins is initiated in the ER. Plant J. 62, 601–614 (2010).

    Article  CAS  Google Scholar 

  28. Nishikawa, S., Hirata, A. & Nakano, A. Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies. Mol. Biol. Cell 5, 1129–1143 (1994).

    Article  CAS  Google Scholar 

  29. Tse, Y. C. et al. Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell 16, 672–693 (2004).

    Article  CAS  Google Scholar 

  30. Humair, D., Hernandez Felipe, D., Neuhaus, J. M. & Paris, N. Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor. Plant Cell 13, 781–792 (2001).

    Article  CAS  Google Scholar 

  31. Runions, J., Brach, T., Kuhner, S. & Hawes, C. Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane. J. Exp. Bot. 57, 43–50 (2006).

    Article  CAS  Google Scholar 

  32. daSilva, L. L. et al. Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting. Plant Cell 17, 132–148 (2005).

    Article  CAS  Google Scholar 

  33. Pimpl, P. et al. Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway. Plant Cell 18, 198–211 (2006).

    Article  CAS  Google Scholar 

  34. Scheuring, D. et al. Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway. BMC Plant Biol. 12, 164–180 (2012).

    Article  CAS  Google Scholar 

  35. Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).

    Article  CAS  Google Scholar 

  36. Rocchetti, A., Hawes, C. & Kriechbaumer, V. Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody. Plant Methods 10, 12 (2014).

    Article  Google Scholar 

  37. Schornack, S. et al. Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60, 744–754 (2009).

    Article  CAS  Google Scholar 

  38. Watanabe, E. et al. An ER-localized form of PV72, a seed-specific vacuolar sorting receptor, interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves. Plant Cell Physiol. 45, 9–17 (2004).

    Article  CAS  Google Scholar 

  39. Hillmer, S., Movafeghi, A., Robinson, D. G. & Hinz, G. Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus. J. Cell Biol. 152, 41–50 (2001).

    Article  CAS  Google Scholar 

  40. Hinz, G., Colanesi, S., Hillmer, S., Rogers, J. C. & Robinson, D. G. Localization of vacuolar transport receptors and cargo proteins in the Golgi apparatus of developing Arabidopsis embryos. Traffic 8, 1452–1464 (2007).

    Article  CAS  Google Scholar 

  41. Viotti, C. et al. Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22, 1344–1357 (2010).

    Article  CAS  Google Scholar 

  42. Wang, H., Zhuang, X., Hillmer, S., Robinson, D. G. & Jiang, L. Vacuolar sorting receptor (VSR) proteins reach the plasma membrane in germinating pollen tubes. Mol. Plant 4, 845–853 (2011).

    Article  CAS  Google Scholar 

  43. Schmitt, F. J. et al. eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM). Biochim. Biophys. Acta 1837, 1581–1593 (2014).

    Article  CAS  Google Scholar 

  44. Watanabe, E., Shimada, T., Kuroyanagi, M., Nishimura, M. & Hara-Nishimura, I. Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin. J. Biol. Chem. 277, 8708–8715 (2002).

    Article  CAS  Google Scholar 

  45. Stael, S. et al. Plant organellar calcium signalling: an emerging field. J. Exp. Bot. 63, 1525–1542 (2012).

    Article  CAS  Google Scholar 

  46. Ordenes, V. R. et al. In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics. Cell Calcium 52, 397–404 (2012).

    Article  CAS  Google Scholar 

  47. Etxeberria, E., Gonzalez, P., Baroja-Fernandez, E. & Romero, J. P. Fluid phase endocytic uptake of artificial nano-spheres and fluorescent quantum dots by sycamore cultured cells: evidence for the distribution of solutes to different intracellular compartments. Plant Signal Behav. 1, 196–200 (2006).

    Article  Google Scholar 

  48. Phillipson, B. A. et al. Secretory bulk flow of soluble proteins is efficient and COPII dependent. Plant Cell 13, 2005–2020 (2001).

    Article  CAS  Google Scholar 

  49. Bubeck, J. et al. The syntaxins SYP31 and SYP81 control ER-Golgi trafficking in the plant secretory pathway. Traffic 9, 1629–1652 (2008).

    Article  CAS  Google Scholar 

  50. Pimpl, P., Hanton, S. L., Taylor, J. P., Pinto-DaSilva, L. L. & Denecke, J. The GTPase ARF1p controls the sequence-specific vacuolar sorting route to the lytic vacuole. Plant Cell 15, 1242–1256 (2003).

    Article  CAS  Google Scholar 

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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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  1. Centre for Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 32, D-72076 Tübingen, Germany

    Fabian Künzl, Simone Früholz, Florian Fäßler, Beibei Li & Peter Pimpl

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  1. Fabian Künzl
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Contributions

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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Correspondence to Peter Pimpl.

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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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