Faculté des sciences

The role of palmitoylation in the secretoy pathway of plants

Stigliano, Egidio ; Neuhaus, Jean-Marc (Dir.)

Thèse de doctorat : Université de Neuchâtel, 2011.

This study aimed to study an important eukaryotic post-translational modification, the S-palmitoylation. Until now, there was no study of palmitoylation in plant cell biology. In the first part of this study, we wanted to study the palmitoylation of the vacuolar receptor AtRMR1, as predicted in silico. We used a very innovative technique, the Biotin Switch Assay, which does not use... More

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    Summary
    This study aimed to study an important eukaryotic post-translational modification, the S-palmitoylation. Until now, there was no study of palmitoylation in plant cell biology. In the first part of this study, we wanted to study the palmitoylation of the vacuolar receptor AtRMR1, as predicted in silico. We used a very innovative technique, the Biotin Switch Assay, which does not use radioactive palmitate, is much less time-consuming as it is possible to obtain results after three to four day. Another advantage for cell biology is that it allows the characterization of entire palmitoyl-proteomes. Yeast and neuronal palmitoyl-proteomes have indeed been recently characterized. The study of RMR1’s palmitoylation revealed the first palmitoylated plant transmembrane protein. The palmitoylation of a small fraction of RMR1 at a higher molecular weight deserves further discussion.
    In the second part of this thesis, I addressed the more general role of palmitoylation in the secretory pathway through the use of a potent palmitoylation inhibitor: 2BP. This study showed a specific action of the drug in TGN/post-TGN compartments. The drug affected the structural maintenance of macrovesicles of secretion. The macrovesicles of secretion have recently been characterized by cryofixation and by electron tomography. They are structures reminiscent of a bunch of grapes, where each grape is a secretory vesicle. They are associated with the building with TGN-rich secretory vesicles with a diameter of few tens of nanometres. These vesicles are particularly visible in tissues with a high growth rate, such as pollen tubes. 2BP drastically changed the state of aggregation of macrovesicles of secretion. In an imaged way, each grape was released and a diffuse fluorescence was observed. Palmitoylation is therefore important in the formation or stability of this important secretory structure. A possible extension of this work would be the isolation of palmitoylated proteins involved in this stabilization. In addition, palmitoylated Rabs were detected for the first time in a plant. The three plant Rabs for which I detected the effect of 2BP are located in post-Golgi compartments.
    In a last part of the thesis, I decided to investigate the route of secretion of GFP-Chi, a vacuolar markerwidely used in the lab that can be followed along the route of secretion. Apparent contradictions have been reported: a vacuolar sorting of this marker by the Golgi-TGN-PVC pathway or an independent-COPII trafficking that can bypass the classic route. Unexpectedly, when GFP-Chi was co-expressed with NtSar1H74L (a dominant-negative mutant blocking the ER-Golgi trafficking by preventing the formation of COPII vesicles), the reporter reached the vacuole. This suggests that the alternative pathway bypassing the Golgi can take place. I also detected the presence of a possible GFP-Chi dimer associated with the membrane fraction upon ultracentrifugation. The nature of this dimer of a soluble protein remains to be investigated although several cases of aggregation of soluble proteins have been reported in the literature (like β-amyloids presents in the of Alzheimer disease). Another matter of great interest is whether the dimer reaches the vacuole or is only transient intermediate during the transport to the vacuole.