Analysis of the Inheritance and Positioning of the Golgi Apparatus

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9604342 Glick The Golgi apparatus has a distinctive structure and protein composition, but the processes that give this organelle its unique character are only beginning to be elucidated. For example, it is still unclear how Golgi structures are inherited during cell division or how the Golgi is localized to a particular region of the cytoplasm. A key to understanding these phenomena comes from the finding that Golgi stacks are often situated near vesiculating "transitional" regions of the endoplasmic reticulum (ER). The working hypothesis for this project is that Golgi stacks are located near transitional ER sites because Golgi assembly occurs at these sites, with new Golgi cisternae forming by the coalescence of ER-derived vesicles. In this view, the Golgi can be considered a dynamic outgrowth of the ER. The ultimate goal of this work is to determine how a cell creates and propagates the transitional ER subdomains that give rise to Golgi stacks. The experimental approach is to analyze Golgi inheritance and positioning in two budding yeasts, Saccharomyces cerevisiae and Pichia pastoris. In S. cerevisiae, the Golgi has a fragmented structure and rarely displays a stacked cisternal morphology. Nevertheless, it is possible to study Golgi inheritance in S. cerevisiae by monitoring the movement of Golgi elements into the bud. For studies of Golgi positioning, P. pastoris is the organism of choice because it contains a small number of discrete Golgi stacks that resemble the organelles found in higher eukaryotes. Golgi stacks in P. pastoris are located next to vesiculating membranes that evidently correspond to transitional ER. Because P. pastoris is closely related to S. cerevisiae and can be genetically manipulated in much the same way, it will be valuable to perform a comparative study of the Golgi in these two yeasts. The project has two Specific Aims. The first is to analyze the mechanism by which proteins are localized to the transitional ER in P . pastoris. Antibodies are being raised against P. pastoris components of the COPII- coated vesicle system, which defines the transitional ER. Immunolocalization experiments will be performed to confirm that these proteins are restricted to discrete subdomains of the ER. Based on functional studies in S. cerevisiae, it seems likely that the specific recruitment of COPII components to the transitional ER depends upon a restricted localization of Sec12p, the protein that initiates the COPII assembly pathway. This prediction will be tested by modifying the P. pastoris SEC12 gene in an attempt to alter its localization. In addition, affinity methods will be used to isolate partner proteins that may anchor Sec12p in transitional ER zones. The second aim is to carry out genetic screens for identifying proteins that mediate Golgi inheritance in S. cerevisiae. A strain of yeast has been created in which Golgi organelles are labeled with Green Fluorescent Protein (GFP). These cells will be mutagenized and screened visually to identify mutants that show defects in Golgi inheritance. The relationship between ER and Golgi elements will be examined in the mutant cells, and the genes responsible for the mutant phenotypes will be cloned and characterized. The Golgi apparatus is a complex organelle of eukaryotic cells which consists of a highly specialized collection of membrane-enclosed compartments in which newly synthesized proteins and certain complex glycoconjugates are covalently modified and other complex glycoconjugates are synthesized. Most of the proteins and complex glycoconjugates within the Golgi complex, whether soluble inside the compartments or membrane-bound, are only transiently resident there; once the syntheses and modifications are completed, these macromolecules are sorted and transported to other sites inside or outside the cell, where they carry out their specific functions. The Golgi complex may assume different morphologies and subcellular locations (and even specific metabolic functions) depending on the specific cell type and the stage of the life cycle of the cell. Although the Golgi complex has been studied intensively for decades, and much is known about specific Golgi enzymes and the fate of macromolecules that pass through the complex, there is still much about it that remains poorly understood. This project addresses fundamental questions about the biogenesis of the Golgi complex, utilizing cutting-edge technologies and approaches. If the results support Dr. Glick's hypothesis, they will force a change in the way investigators think about the Golgi complex, in terms of its function and its origin within the cell. ***