Research objectives and content
The major focus of this proposal is to study the mechanisms of protein transport through the Golgi compartments in yeast. Based on previous studies, transport through the Golgi is thought to be SNARE-mediated. Two v-SNAREs (Sft1p and p28) are known in the yeast Golgi. Moreover, two candidates t-SNAREs (tsx and tsz) have been identified by homology searches and are located in the Trans Golgi Network. We will investigate the role of these proteins in the intra-Golgi anterograde transport using genetic approach and various in vivo functional assays. The oartners of tsx/tsz in SNARE complexes (Sft1p, p28 or others) will be identified. A related study will consist in the characterization of the nature of connections between Golgi compartments. We will try to observe budding and fusion events directly in vivi using the Green Fluorescent Protein technology. Moreover, a cell free functional assay for intra-Golgi transport will be developped. These two methods will be very helpful to determine whether intra-Golgi anterograde traffic is vesicle-mediated and to analyse in detail the biochemical requirements of these transport processes.