Characterization of Saccharomyces cerevisiae trans Golgi network protein trafficking events using cell-free systems

The regulated transfer of protein and lipid cargo between organelles is essential to the maintenance of eukaryotic cell structure. Such processes are particularly critical at the trans Golgi network (TGN), where extensive, highly coordinated protein sorting must occur to mediate vesicular communication between the TGN and other organelles including the vacuole, endosomes, the plasma membrane and earlier Golgi compartments. To gain a better understanding of protein trafficking at the TGN, we use the baker's yeast, Saccharomyces cerevisiae as a model system to study the transmembrane TGN resident processing protease, Kex2p. Kex2p maintains steady-state localization to the TGN by cycles of transport between TGN and endocytic compartments, making it an excellent reporter for vesicular delivery between these compartments. Using Kex2p as a marker, we have begun to reconstitute TGN transport and fusion events in cell-free systems.; Initial experiments identified a previously uncharacterized membrane fusion event, homotypic fusion of TGN compartments. Homotypic TGN fusion was found to require a t-SNARE complex consisting of Tlg2p, Tlg1p and Vti1p. Fusion was impaired between TGN membranes lacking function of the rab GTPase, Vps21p. A putative membrane tethering complex protein Pep3p was also required for fusion. As is the case for homotypic fusion of other endomembrane compartments, this fusion likely reflects a basic mechanism for maintenance of the integrity of the TGN.; To further the analysis of Kex2p trafficking at the TGN, we developed a cell-free assay for the specific transport of Kex2p from the TGN to the late endosome/prevacuolar compartment (PVC). This transport step is critical for the biogenesis of the yeast vacuole/lysosome as it is the step in which hydrolytic enzyme precursors are sorted away from secretory protein traffic for delivery into the endosomal/lysosomal membrane system. Cell-free TGN to PVC transport requires Pep12p, a neuronal syntaxin homolog thought to mediate fusion of all incoming vesicular traffic at the prevacuolar compartment.; This work enhances our understanding of TGN trafficking by illuminating a novel membrane fusion event, homotypic TGN fusion, that may be important for TGN maintenance and sorting, and sets the stage for biochemical dissection of TGN to late endosomal delivery of lysosomal precursor enzymes.