Dynamics of the localization of the glycosylphosphatidylinositol-containing protein Gas1 during the yeast life cycle

Gas proteins are beta-(1,3)-glucan elongases which play a crucial role in the assembly and remodeling of the cell wall at different stages of the yeast life cycle. Gas1 is the best characterized member out of a family of five (Gas1 to Gas5). GAS1 gene is actively expressed during vegetative growth and its loss causes a round cell shape, incomplete bud growth, defects in cell separation and modifications in the structure of the cell wall. In this work we have used two different fusions of Gas1p to fluorescent proteins, RFP-Gas1p and Gas1p-GFP, to investigate Gas1p localization during the entire yeast life cycle. In vegetative growth Gas1p was detected at the cell periphery, in the bud neck and in the bud scars. The fluorescence in the plasma membrane was more intense at discrete sites indicating that Gas1p is sequestered in lipid rafts. This was confirmed by a floatation assays indicating that Gas1p-GFP is recovered in the DRMs fraction as wild type Gas1p. The Gas1p associated to lipid rafts was subjected to protein turnover and represented a mobile pool of molecules. Another pool of Gas1p was immobile and consisted in the molecules which were stably bound to the chitin ring and bud scars. At the bud scar Gas1p-GFP remained fluorescent for several generations. In a chs3 null mutant the lack of the chitin ring and of the Gas1p cross-linked to it unveiled a further localization of Gas1p along the septum line in cells at cytokinesis. Thus Gas1p is also localized to the primary septum. In the chs1, chs2 and chs3 single null mutant Gas1p was released into the growth medium indicating that Gas1p is linked to all type of chitin synthesized in the cell. Finally we studied the destiny of Gas1p during the process of meiosis and sporulation and during germination. GAS1 gene expression is repressed during sporulation but the protein persists for many days. Interestingly Gas1p is translocated from the plasma membrane to the prospore membrane by an endocytotic pathway which partially relies on the END3 gene product. Moreover, our results indicate that Gas1p is required during the polarized growth of the germinating spore. Acknowledgement: RTN project Cantrain N. 51248 to LP