Purification, biosynthesis and cellular localization of a major 125‐kDa glycophosphatidylinositol‐anchored membrane glycoprotein of Saccharomyces cerevisiae
- 1 January 1991
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 195 (2), 439-448
- https://doi.org/10.1111/j.1432-1033.1991.tb15723.x
Abstract
The yeast Saccharomyces cerevisiae has been shown to contain a major 125-kDa membrane glycoprotein which is anchored in the lipid bilayer by a glycophosphatidylinositol anchor. This protein was purified to near homogeneity and was used to raise a rabbit antibody. Biosynthesis of the 125-kDa protein was studied by immunoprecipitation of 35SO4-labeled material from wild-type cells or a secretion mutant (sec18) in which the vesicular traffic from the endoplasmic reticulum (ER) to the Golgi is blocked. The 125-kDa protein is first made in the ER as a 105-kDa precursor which already contains a glycophosphatidylinositol anchor and which is slowly transformed into the 125-kDa form upon chase (t1/2 approximately 10-15 min). The 105-kDa precursor can be reduced to an 83-kDa form by the enzymatic removal of N-glycans. The removal of N-glycans from the mature 125-kDa protein yields a 95-kDa species. Thus, removal of the N-glycans does not reduce the ER and mature forms to the same molecular mass, indicating that not only elongation of N-glycans but also another post-translational modification takes place during maturation. Selective tagging of surface proteins by treatment of 35SO4-labeled cells with trinitrobenzene sulfonic acid at 0 C followed by immunoprecipitation of the tagged proteins shows that the 125-kDa protein, but not the 105-kDa precursor, becomes transported to the cell surface. This tagging of cells after various lengths of chase also shows that the surface appearance of the protein is biphasic with about one half of the mature 125-kDa protein remaining intracellular for over 2 h. Glycosylation and/or glycophosphatidylinositol anchor addition is important for the stability of the 125-kDa protein since the protein remains undetectable in sec53, a temperature-sensitive mutant which does not make GDP-mannose at 37 C and does not add glycophosphatidylinositol anchors at 37 degrees C.Keywords
This publication has 42 references indexed in Scilit:
- Detection of an intermediate compartment involved in transport of alpha-factor from the plasma membrane to the vacuole in yeast.The Journal of cell biology, 1990
- CELL-SURFACE ANCHORING OF PROTEINS VIA GLYCOSYL-PHOSPHATIDYLINOSITOL STRUCTURESAnnual Review of Biochemistry, 1988
- Complete structure of the glycosyl phosphatidylinositol membrane anchor of rat brain Thy-1 glycoproteinNature, 1988
- Structural and Functional Roles of Glycosyl-Phosphatidylinositol in MembranesScience, 1988
- Protein glycosylation in yeastBiochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, 1987
- Eukaryotic protein modification and membrane attachment via phosphatidylinositolCell, 1987
- Export of major cell surface proteins is blocked in yeast secretory mutantsThe Journal of cell biology, 1983
- Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeastCell, 1981
- The Role of Dolicholmonophosphate in Glycoprotein Biosynthesis in Saccharomyces cerevisiaeEuropean Journal of Biochemistry, 1974
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970