96-31129 Waters The secretory pathway of eukaryotic cells consists of a set of membrane-bounded compartments that are involved in delivery of newly synthesized proteins to their final destinations. The Golgi apparatus is involved in the post-translational modification and sorting of proteins that pass through it. The Golgi consists of several topologically distinct cisternae, each contains a unique set of resident proteins. These proteins resist dispersion by the large flux of vesicular traffic to and from the Golgi apparatus. The maintenance of the Golgi integrity is an essential process, yet it is poorly understood. Do newly synthesized Golgi proteins simply cease their forward movement upon reaching the correct cisternae, or do they localized by a more dynamic mechanism involving retrieval from distal compartments? The goal of this proposal is to gain insights into Golgi retention mechanisms. The budding yeast Saccharomyces cerevisiae is used as a model system since it is amenable to biochemical, cell biological, and genetic analyses. The Golgi mannosyltransferase, Och1p, will serve as the model protein because the inability to retain Och1p in the Golgi is readily detectable by both biochemical and genetic techniques. Fusion proteins of Och1p fused to the reporter protein invertase have been used in initial studies. Inclusion of a cleavage site specific for a late Golgi endoprotease, Kex2p, in some of the fusion proteins, allows monitoring of the release of the invertase moiety if the fusion protein reaches the distal compartments of the Golgi. Initial results from these studies imply that newly synthesized Och1p is not retained efficiently on its first passage through the Golgi apparatus, that is, Och1p moves to distal compartments and is retrieved. Retention in the Golgi apparatus is not a static process, but is dynamic, involving, apparently, both anterograde and retrograde vesicular transport. The mechanism of this process will be further investigated by identifying components o f the cellular machinery that retains Och1p and mediates its retrieval from distal compartments; by determining if Och1p recycles through the ER and the endosomes, as well as the Golgi, by using mutants that can "trap" recycling Och1p in the ER or divert it from the endosomes to the plasma membrane; and by biochemically characterizing the statically-retained form of Och1p. Known mutants of the retrograde vesicular traffic will be examined to study their impact on the recycling of Och1p through the Golgi. New components of the retrograde transport apparatus will be identified by selecting mutants defective in recovering Och1p from distal compartments. These mutants will be phenotypically characterized and the gene will be examined at the molecular level. Studying protein retention in the Golgi of S. cerevisiae will be relevant to Golgi retention processes in mammalian cells as well as to the maintenance of other organelles. Since there exist numerous examples of evolutionary conservation of biological processes among various species, these studies will help reveal how human cells maintain compartmentalization that is so critical to cell viability. %%% The secretory pathway of eukaryotic cells consists of a set of membrane-bounded compartments that are involved in delivery of newly synthesized proteins to their final destinations. The Golgi apparatus is involved in the post-translational modification and sorting of proteins that pass through it. The Golgi consists of several topologically distinct cisternae, each contains a unique set of resident proteins. These proteins resist dispersion by the large flux of vesicular traffic to and from the Golgi apparatus. The maintenance of the Golgi integrity is an essential process, yet it is poorly understood. Do newly synthesized Golgi proteins simply cease their forward movement upon reaching the correct cisternae, or do they localized by a more dynamic mechanism involving retrieval from distal compartments? The goal of this proposal is to gain insi ghts into Golgi retention mechanisms. Studying protein retention in the Golgi of S. cerevisiae will be relevant to Golgi retention processes in mammalian cells as well as to the maintenance of other organelles. Since there exist numerous examples of evolutionary conservation of biological processes among various species, these studies will help reveal how human cells maintain compartmentalization that is so critical to cell viability. ***
