The plant early secretory pathway : molecular tools to study endoplasmic reticulum to golgi apparatus transport

Eukaryotic cells contain several membrane-bound organelles, some of which are collectively referred to as the endomembrane system. Proteins and other molecules travel from one subcellular compartment to an other thanks to the secretory pathway. The journey from the endoplasmic reticulum (ER) to the Golgi apparatus is regulated by the Coat Protein complex II (COPII). Although COPII proteins are conserved among all eukaryotic organisms, the plant secretory pathway exhibits specific features that are not present in Saccharomyces cerevisiae or animals cells, and the molecular mechanisms of protein sorting remain partially unknown. In this work, we developed a biochemical assay able to reconstitute, in vitro, the budding of COPII vesicles from ER membranes isolated from Arabidopsis thaliana cells. This transfer was energy dependent, and required a COPII proteins source, provided by a cytosolic extract. In order to optimize the in vitro assay, we tried to purify a complete set of plant COPII proteins, to replace the cytosolic fraction. We expressed these proteins in Escherichia coli and S. cerevisiae and purified them by affinity chromatography. Unfortunately, four out of the five COPII proteins appeared to be insoluble, preventing their purification. In parallel to the biochemical approach, we generated yeast strains expressing plant COPII proteins as a replacement for yeast counterparts. For instance, we replaced the yeast ScSEC24 gene by a sequence coding for an hexa-histidine tagged version of AtSec24. We expressed plant plasma- membrane proteins in this strain and investigated their transport.(AGRO 3) -- UCL, 201