LOCALIZATION AND CELL-SURFACE ANCHORING OF THE SACCHAROMYCES-CEREVISIAE FLOCCULATION PROTEIN FLO1P

The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-aci d serine- and threonine-rich protein involved in flocculation. We have assessed the localization of Flo1p by immunoelectron microscopy, and in this study we show that this protein is located in the external man noprotein layer of the cell wall, at the plasma membrane level and in the periplasm, The protein was also visualized in the endoplasmic reti culum and in the nuclear envelope, indicating that it was secreted thr ough the secretory pathway, The protein was detected by Western blotti ng in cell wall extracts as a high-molecular-mass (>200 kDa) polydispe rse material obviously as a result of extensive N and probably O glyco sylation, Flo1p was extracted from cell walls in large amounts by boil ing in sodium dodecyl sulfate, suggesting that it is noncovalently anc hored to the cell wall network The membranous forms of Flo1p were show n to be solubilized by phosphatidylinositol-phospholipase C treatment, suggesting that Flo1p is glycosyl phosphatidylinositol (GPI) anchored to this organelle. The expression of truncated forms with the hydroph obic C-terminal domain deleted led to the secretion of the protein in the culture medium, The hydrophobic C terminus, which is a putative GP I anchoring domain, is therefore necessary for the attachment of Flo1p in the cell wall, Deletion analysis also revealed that the N-terminal domain of Flo1p was essential for cellular aggregation, On the whole, our data indicate that Flo1p is a true cell wall protein which plays a direct role in cell-cell interaction.