Die Rolle der Exocyst-Untereinheiten Sec6 und Sec8 im apikalen Transport der Saccharase-Isomaltase

Epithelial cells are able to form and sustain different functional areas in their plasma membrane. The apical membrane faces the lumen (environment) and the basolateral membrane faces the basement membrane and neighbouring cells. The two membrane domains differ in their protein and lipid composition. These different types of membranes are separated by tight junctions and sustained by a selective transport. Different sorting signals are important for the selective transport and for the sorting of proteins in respective transport vesicles. For the basolateral transport specific amino acid sequences in the cytoplasmic part of the protein are important. Apical sequences are more complex and consist of N- and O-glycans, GPI-anchors, and transmembrane domains. These apical sorting signals bias if apical-localized and -secreted proteins take the lipid raft associated or the non-lipid raft associated route to the apical membrane. Lipid rafts are special membrane microdomains within cell membranes. Transport vesicles with their selective cargo bind to the target membrane on the route to the cell surface before fusion occurs. Vesicle binding to the target membrane is arranged by tethering complexes. One of these tethering complexes is the so-called “Exocyst” complex, a multiprotein complex consisting of eight subunits. Exocyst subunits can be found on the transport vesicle (ligand) as well as on the target membrane (receptor). Ligand and receptor arrange the binding before fusion occurs. The fusion of the vesicle and the membrane is then triggered by SNARE-complexes. In polarized MDCK cells (kidney epithelial cells) the main part of the Exocyst complex is located on the lateral membrane near the tight junctions. A decisive role of the Exocyst complex in protein trafficking could previously be shown for some basolateral localized proteins in MDCK cells. Within this dissertation, the role of some Exocyst subunits, especially for Sec8, in the apical transport of the model protein sucrase-isomaltase (SI) in MDCK cells were investigated. The starting point was the identification of the Exocyst subunit Sec6 on SI carrying, apical transport vesicles twenty minutes after the release from trans-Golgi network in polarized MDCK-SI-YFP cells. Immunoprecipitation experiments could show an association of another Exocyst subunit, Sec8, with SI carrying vesicles. Sec8 interacts indirectly with SI. Reduction of endogenous Sec8 concentration by specific siRNA shows no effect on the apical transport of SI in MDCK-SI cells. Microscopic investigations in MDCK-SI-YFP cells suggest an interaction of Sec6 and Sec8 with SI carrying vesicles on the lateral membrane. This membrane area corresponds to the main localization of the Exocyst complex in polarized MDCK cells and resides near the tight junctions. Microscopic investigations in unpolarized COS cells (fibroblasts) shows no colocalization of SI carrying vesicles with the Exocyst subunits Sec6 and Sec8. Sec6 and Sec8 are localized in COS cells predominantly on recycling endosomes. SI is transported on a lipid raft-dependent route to the apical membrane. Isolation of these special membrane microdomains shows that the subunit Sec8 is also associated with detergent resistant membranes (DRMs) in MDCK-SI cells. The data obtained within this dissertation suggest, that the Exocyst subunit Sec8 has no influence on the apical transport of SI in MDCK cells. At this time, no conclusion can made where the association of Sec8 with DRMs occurs. Further investigations are necessary in this regard.