Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p.
The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-acid 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 mannoprotein layer of the cell wall, at the plasma membrane level and in the periplasm. The protein was also visualized in the endoplasmic reticulum and in the nuclear envelope, indicating that it was secreted through the secretory pathway. The protein was detected by Western blotting in cell wall extracts as a high-molecular-mass (>200 kDa) polydisperse material obviously as a result of extensive N and probably O glycosylation. Flo1p was extracted from cell walls in large amounts by boiling in sodium dodecyl sulfate, suggesting that it is noncovalently anchored to the cell wall network. The membranous forms of Flo1p were shown 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 hydrophobic C-terminal domain deleted led to the secretion of the protein in the culture medium. The hydrophobic C terminus, which is a putative GPI 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.
Bony
M
M
Laboratoire de Microbiologie et Technologie des Fermentations, IPV, INRA-ENSA, Montpellier, France.
Thines-Sempoux
D
D
Barre
P
P
Blondin
B
B
D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions; D010738:Type C Phospholipases / Q000378:metabolism
C087578:FLO1 protein, S cerevisiae; D005656:Fungal Proteins; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase