The role of the AP -1 adaptor complex in trafficking between the trans -Golgi network and endosomal system.

摘要

In Sacchromyces cerevisiae it is generally accepted that there are two routes for trafficking of proteins from the trans-Golgi network (TGN) to the vacuole. One involves direct transport from the TGN to the vacuole. The second involves transport from the TGN to the prevacuolar compartment (PVC) via GGA coated vesicles, followed by PVC to vacuole transport. We propose that there is an alternative third route. This route entails transit from the TGN to the early endosome (EE), followed by delivery to the PVC and subsequent transit to the vacuole.;To test this hypothesis, the processing kinetics of the protein A(F→A)-ALP was examined. Its processing only occurs in the vacuole. Here it is shown that processing of A(F→A)-ALP is contingent upon delivery to the PVC. Processing is blocked in strains lacking functional Pep12p, a PVC t-SNARE required for vesicle docking at the PVC. In support of an alternative route, the processing kinetics of A(F→A)-ALP is not affected by mutations in the GGA proteins. This is in contrast to other proteins that use the GGA pathway, as their delivery to the vacuole is significantly slowed when GGA function is ablated. Further support of an EE itinerary is the observation that A(F→A)-ALP colocalizes with the lipophilic dye, FM4-64 at a time when the dye is predominantly associated with the EE. Disruption of the AP-1 vesicle coat complex leads to an accelerated processing of A(F→A)-ALP. Additionally, a pull down assay reveals that there is a physical interaction between two of the four AP subunits with A(F→A)-ALP. Deletion of this region results in accelerated processing time of A(F→A)-ALP.;Appending the region of A(F→A)-ALP that interacts with both subunits of AP-1 to Cps1p, a protein that does not normally transit through the EE delays its progress to the vacuole when it is forced to use the TGN-EE-PVC pathway. These results are consistent with a model in which A(F→A)-ALP traffics through the EE in transit to the vacuole. It physically interacts with AP-1, and this interaction delays its delivery to the vacuole. In mammalian systems, AP-1 has been implicated in transport from the EE to the TGN. Data presented in this thesis are consistent with this model and suggests that in Sacchromyces cerevisiae AP-1 functions as a retrieval mechanism from the EE to the TGN.