Laboratory of Biochemistry and Molecular Biology

Targeting of Vacuolar Proteins in Plants
Back to main page of: Lab of biochemistry, Institute of Botany, Biology, University of Neuchâtel

Our main interest is in the mechanisms of protein sorting and transport from the endoplasmic reticulum into vacuoles . Recently, we (and many other plant biochemists) realized that plant cells have a complex vacuolar system, as it could be shown that some cells can contain at least two functionally distinct vacuoles; one with lytic function and the other that could be a storage compartment. In fact, there may be at least three, in a complex arrangement as observed in seeds (Jiang, L.W.et al., 2000, J. Cell Biol. 150, 755-769). This explains why there are several types of vacuolar sorting determinants (VSD) (#5 and 6).

We have characterized a VSD in the precursor of a vacuolar protein, the tobacco chitinase A (#1). This VSD is a C-terminal propeptide. We have also contributed to the characterization of the C-terminal VSD of phaseolin (#11) and of the internal sequence-specific VSD of ricin (#14). We are now looking for the sorting receptor for C-terminal VSDs, as well as for other components involved in the sorting process. We are using the Green Fluorescent Protein (GFP) as a reporter protein. We can visualize a subset of pH-neutral vacuoles that accumulate the GFP fused to the VSD of chitinase A (#4), and another subset of more acidic vacuoles that accumulate the GFP fused to the VSD of barley aleurain (#15).

We are also investigating the processes of vacuolar sorting to the lytic compartment using the vacuolar sorting receptor (VSR) from pea, which was identified as binding the sequence-specific VSD from the barley protease, aleurain (# 2, 5 and 7). In parallel we would like to characterize further the compartments involved in these processes.

We could show (for the first time in vivo), in yeast, the interaction between the VSD of aleurain (in fact, a homologue from Petunia) and the vacuolar sorting receptor VSR-PS1 from pea (#13).

The influence of the transmembrane segment of class I proteins on intracellular trafficking was investigated. It appears that in the absence of a cytosolic tail, these proteins will accumulate in the ER, the Golgi or the plasmalemma, depending on the length of the transmembrane segment (the longer, the further the protein will get. No such protein accumulated in a tonoplast, excluding these membranes from a "default" pathway (#16).

Publications
Links to other plant labs involved in related research

STAFF

 

From left: Jeannine Okmeni Nguemélieu (PhD student), Olivier Zava (PhD student), Doramys Hernández Felipe (PhD student), Jean-Marc Neuhaus (head of the group), Ilian Simidjiev (post-doc)

Sophie Marc-Martin is our technician, a biologist with a vast experience in molecular biology. She is making most of the constructions and a lot of the work with protoplasts. She is also our sequencing expert.

Ricardo Flückiger is a PhD student working with Arabidopsis expressing the two vacuolar GFPs developed by Gian-Pietro Di Sansebastiano.

We collaborate with several other laboratories, some colleagues being former collaborators or students:

Nadine Paris came to Neuchâtel as a post-doctoral fellow and brought along the clones of BP-80/VSR she had obtained in John Rogers' lab, then in Saint-Louis 
She studies the sorting of the vacuolar sorting receptors and the trafficking of membrane proteins in plants.

She now works in Rouen as a CNRS fellow (Nadine.Paris @ univ-rouen.fr)

Gian-Pietro Di Sansebastiano  worked with protoplasts, cell suspensions and transgenic tobacco and Arabidopsis plants. He introduced the two different soluble vacuolar GFPs as markers of the secretory pathway (#8, 14).

After working as a post-doc in Wye college (England), Gipo moved back to his home town, the beautiful city of Lecce (Apulia, Italy). He works on the process of secretion in plant cells.(Gp.disansebastiano @ unile.it)
Susannah Gal from the State University of New York in Binghampton (email: sgal @ binghamton.edu) spent a sabbatical year in our laboratory. She works on vacuolar proteases in plants. We are part of a European Research Training Network on "Biochemical and genetic approaches to study bio-molecular interactions in plants" along with six other laboratories

Recent own publications on the subject:

  1. Neuhaus, J.-M., Pietrzak, M. and Boller, T.  (1994) Mutation analysis of the C-terminal vacuolar targeting peptide of tobacco chitinase: low specificity of the sorting system, and gradual transition between intracellular retention and secretion into the extracellular space. Plant Journal 5, 45-54.
  2. Freydl, E., Meins, F., Jr. , Boller, T. and Neuhaus, J.-M. (1995) Kinetics of prolyl hydroxylation, intracellular transport and C-terminal processing of the tobacco vacuolar chitinase. Planta 147, 250-256.
  3. Neuhaus, J.-M. (1996) Protein targeting to the plant vacuole. Plant Physiol. Biochem. 34, 217-221.
  4. Di Sansebastiano, G.-P., Paris, N., Marc-Martin, S. and Neuhaus, J.-M. (1998). Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway. Plant Journal 15, 449-457
  5. Neuhaus, J.-M. and Rogers, J.C. (1998). Sorting of proteins to vacuoles in plant cells. Plant Molecular Biology 38, 127-144 (pdf, Copyright belongs to Kluwer)
  6. Matsuoka, K. and Neuhaus, J.-M. (1999). cis-elements of protein transport to the plant vacuoles. J. Experimental Botany 50, 165-174
  7. Neuhaus, J.-M. and Martinoia, E (1999). Plant cell vacuoles. Encyclopedia of Life Sciences. in (electronic) press
  8. Grieco F., Castellano M.A., Di Sansebastiano G-P., Maggipinto G., Neuhaus J-M. and Martelli G. (1999). Subcellular localization and in vivo identification of olive latent virus 2 putative movement protein. Journal of General Virology, 80, 1103-1109.
  9. Neuhaus, J.-M. (2000). GFP as a marker for vacuoles in plants. In Vacuolar Compartments, D. G. Robinson, and J. C. Rogers, eds. (Sheffield: Sheffield Academic Press Ltd), pp.
  10. Ceriotti A, Paris N, Hillmer S, Frigerio L, Neuhaus J-M, Vitale A, Robinson DG. (2000) Plant cell biology. In Lord, M, Davey, J, eds, Essential cell biology, Oxford University Press, Oxford, pp
  11. Frigerio L, Foresti O, Hernández Felipe D, Paris N, Neuhaus J-M, Vitale A. (2001) The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole. J Plant Physiol 158: 499-503 (pdf, Copyright belongs to the Urban & Fischer Verlag)
  12. Neuhaus, J.-M., and Boevink, P. (2001). The green fluorescent protein (GFP) as a reporter in plant cells. In Plant Cell Biology, C. R. Hawes, and B. Satiat-Jeunemaitre, eds. (Oxford: Oxford University Press), pp. 127-142.
  13. Humair, D., Hernández Felipe, D., Neuhaus, J.-M and Paris, N. (2001) Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor. Plant Cell, 13, 781-792. (pdf, Copyright belongs to the ASPP)
  14. Frigerio L, Jolliffe NA, Di Cola A, Hernández Felipe D, Paris N, Neuhaus J-M, Lord JM, Ceriotti A, Roberts LM. (2001) The internal propeptide of the ricin precursor carries a sequence-specific determinant for vacuolar sorting. Plant Physiol, 126, 167-173 (pdf, Copyright belongs to the ASPP)
  15. Di Sansebastiano, G.-P., Paris, N., Marc-Martin, S., and Neuhaus, J.-M. (2001). Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualised by GFP targeted to either type of vacuoles. Plant Physiol, 126, 78-86 (pdf, Copyright belongs to the ASPP)
  16. Brandizzi, F., Frangne, N., Marc-Martin, S., Hawes, C., Neuhaus, J.-M. and Paris, N. (2002). The Destination for Single-Pass Membrane Proteins Is Influenced Markedly by the Length of the Hydrophobic Domain. Plant Cell, 14, 1077-1092 (pdf, Copyright belongs to the ASPP)
  17. Flückiger, R., De Caroli, M., Piro, G., Dalessandro, G., Neuhaus, J. M., and Di Sansebastiano, G. P. (2003). Vacuolar system distribution in Arabidopsis tissues, visualized using GFP fusion proteins. Journal of Experimental Botany 54, 1577-1584. (pdf, Copyright belongs to Oxford University Press)
  18. Kessler, F., and Neuhaus, J.-M. (2003). Sorting activities in plant cells. Chimia 57, 634-638.
  19. Happel, N., Honing, S., Neuhaus, J. M., Paris, N., Robinson, D. G., and Holstein, S. E. (2004). Arabidopsis micro A-adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR-PS1. Plant J 37, 678-693.(pdf, Copyright belongs to Society for Experimental Biology)

We also follow the work of yeast and animal people. Have a look at the following pages:

GFP Hot Links

Last modifications : 16th September 2004

Jean-Marc Neuhaus