Analyses of vesicular transport within the endoplasmic reticulum-golgi interface in "Saccharomyces cerevisiae"

A characteristic feature of every eukaryotic cell is its division into different compartments. This subdivision into different intracellular organelles like the endoplasmic reticulum (ER), the Golgi apparatus or the endosomal/lysosomal system enables cells to provide the appropriate environment for a great variety of biochemical processes. However, it also necessitates an elaborate machinery for the communication between these compartments or organelles. On one hand, material has to be exchanged between organelles, but on the other hand, their integrity with respect to their protein and lipid content, has to be maintained to fulfil their function. Transport processes between different organelles are mediate by intracellular traffic pathways. Proteins enter the secretory pathway at the ER, where they acquire first posttranslational modifications. From the ER, they are delivered to the Golgi, where they are further modified and sorted to their target compartments. In the secretory pathway, transport carriers, so-called vesicles, bud from one organelle (donor) and fuse with the next organelle (acceptor) along their trafficking route. Understanding the molecular mechanisms and regulations underlying vesicular transport is crucial and therefore has been a main topic of research over the last decades. The machinery required for budding and fusion of vesicles along their trafficking pathways is conserved from yeast to human. Therefore, the yeast Saccharomyces cerevisiae represents a suitable organism to study the secretory pathway.
In this thesis, we used S. cerevisiae to examine the regulation of vesicular traffic at the ER-Golgi interface, more specifically the fusion of vesicles with ER membranes. The consumption of a vesicle at its target membrane is mediated by the orchestrated action of various members of conserved protein families that act in a regulated manner. Main players involved in vesicular fusion are Rab GTPases, tethering factors and SNAREs. The tethering factors and the Rab GTPases mediate the first contact of an incoming vesicle with its acceptor organelle, whereas the SNARE proteins are responsible for the final fusion event between vesicles and target membranes.
Here, we identified the Rab GTPase Ypt1p as mediator of vesicle fusion with the ER. Moreover, Ypt1p was not only required for vesicle fusion at the ER, but also for the maintenance of the morphology and protein composition of the Golgi, and for vesicle formation at the Golgi. In addition, the tethering complex responsible for the docking of Golgi-derived vesicles with the ER, the Dsl1 tethering complex was analyzed. We found that this complex, apart from mediating the first contact of the incoming vesicles with the ER membrane, seems to play an additional role in proofreading and stabilization of SNARE complexes that are responsible for vesicle fusion at the ER