The Golgi complex is involved in the modification of glycoproteins and in the sorting of proteins to their correct destination. Like other subcellular organelles, it contains distinct proteins which are permanent residents and which carry out characteristic biochemical functions. Little is known about the biogenesis of the Golgi complex, arguably the most important organelle in the secretory pathway. Previous studies have clearly demonstrated that different glycosyltransferases are compartmentalized within the different cisternae that make up the Golgi complex. As a result of the spatial organization of these enzymes, nascent secretory proteins undergo progressive and sequential processing as they migrate through the Golgi stacks. Thr proposed experiments aim to identify and characterize yeast mutants which define proteins that function to regulate the spatial organization and activity of resident Golgi proteins. Using the yeast Saccharomyces cerevisiae as an experimental system, a genetic approach will be used to identify factors which regulate Golgi biogenesis. Specifically, experiments are proposed to isolate (a) yeast mutants which fail to properly localize Golgi proteins, thereby defining cellular components which function to mediate Golgi retention and (b) mutants which affect Golgi-specific glycosylation. The rationale behind this latter approach is based on the prediction that secretion and glycosylation are closely coupled and that changes in normal glycosylation may reflect sorting defects within the Golgi. Several mutants with severe glycosylation defects have been identified which also affect other processes that occur int he Golgi. Experiments are proposed to characterize these and other mutants at the genetic, molecular and biochemical level. The use of S. cerevisiae has not been exploited a an experimental system to study the Golgi, although it offers advantages unavailable in other systems. Importantly, it has become clear that there is a high degree of conservation between the secretory pathway of yeast and higher eukaryotes. The long term objective is to understand how Golgi proteins are regulated in mammalian cells. The genetic amenability of yeast will allow the identification and isolation of genes which encode the key players in this problem. Once these yeast genes are isolated, they can be used as molecular probes for their mammalian homologues.
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