scholarly journals Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall.

1995 ◽  
Vol 128 (3) ◽  
pp. 333-340 ◽  
Author(s):  
C F Lu ◽  
R C Montijn ◽  
J L Brown ◽  
F Klis ◽  
J Kurjan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Secretory Pathway ◽  
Molecular Size ◽  
Cross Linking ◽  
Cell Contact ◽  
Gpi Anchor ◽  
Adhesion Protein ◽  
Glycosyl Phosphatidylinositol ◽  
Cross Linkage

The cell adhesion protein alpha-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell-cell contact in mating. alpha-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross-linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-F., J. Kurjan, and P. N. Lipke, 1994. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol. Cell. Biol. 14:4825-4833). Cell wall association of alpha-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against beta 1,6-glucan. Several kre mutants, which have defects in synthesis of cell wall beta 1,6-glucan, had reduced molecular size of cell wall alpha-agglutinin. These findings demonstrate that the cell wall form of alpha-agglutinin is covalently associated with beta 1,6-glucan. The alpha-agglutinin biosynthetic precursors did not react with antibody to beta 1,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOH-terminal truncated form of alpha-agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-beta 1,6-glucan. We propose that extracellular cross-linkage to beta 1,6-glucan mediates covalent association of alpha-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to alpha-agglutinin.

scholarly journals A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin.

1994 ◽  
Vol 14 (7) ◽  
pp. 4825-4833 ◽  
Author(s):  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Soluble Form ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Experimental Support ◽  
Cell Biol ◽  
A Cell

Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall.

A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin

1994 ◽  
Vol 14 (7) ◽  
pp. 4825-4833
Author(s):  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Soluble Form ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Experimental Support ◽  
Cell Biol ◽  
A Cell

Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall.

Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (11) ◽  
pp. 5796-5805
Author(s):  
P Orlean
Keyword(s):  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Nonpermissive Temperature ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Covalent Modifications

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

scholarly journals Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

1997 ◽  
Vol 328 (2) ◽  
pp. 669-675 ◽  
Author(s):  
L. Tamara DOERING ◽  
Randy SCHEKMAN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Proteins ◽  
Attachment Site ◽  
Gpi Anchor ◽  
Mating Pheromone ◽  
Peptide Substrates ◽  
In Vitro System ◽  
Glycosyl Phosphatidylinositol

The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.

scholarly journals Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly.

1990 ◽  
Vol 110 (5) ◽  
pp. 1833-1843 ◽  
Author(s):  
C Boone ◽  
S S Sommer ◽  
A Hensel ◽  
H Bussey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Structural Analysis ◽  
Yeast Cell ◽  
Gene Disruption ◽  
Secretory Pathway ◽  
Yeast Cell Wall ◽  
Beta Glucan ◽  
Polymer Size

The Saccharomyces cerevisiae KRE1 gene encodes a Ser/Thr-rich protein, that is directed into the yeast secretory pathway, where it is highly modified, probably through addition of O-linked mannose residues. Gene disruption of the KRE1 locus leads to a 40% reduced level of cell wall (1----6)-beta-glucan. Structural analysis of the (1----6)-beta-glucan fraction, isolated from a strain with a krel disruption mutation, showed that it had an altered structure with a smaller average polymer size. Mutations in two other loci, KRE5 and KRE6 also lead to a defect in cell wall (1----6)-beta-glucan production and appear to be epistatic to KRE1. These findings outline a possible pathway of assembly of yeast cell wall (1----6)-beta-glucan.

Synthesis of a Glycosylphosphatidylinositol (GPI) fragment as a potential substrate for mannoprotein transglycosidases

Synlett ◽  
2021 ◽  
Author(s):  
Tyson Belz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Drug Development ◽  
Salient Feature ◽  
Cross Linking ◽  
Protective Group ◽  
Fungal Cell ◽  
The Core ◽  
Potential Substrate ◽  
Core Features

A Glycophosphatidylinositol (GPI) tetrasaccharide fragment was synthesized to mimic the core features of premiere model Saccharomyces cerevisiae. The salient feature of this approach is centered on the quick access to different α-1,2 α-1,6-mannosyl and α-1,4-glycosyl linkages using simple glycosylation and protective group techniques. 1D and 2D-J-resolved NMR was used verify the α-configuration for the new linkages. Tetrasaccharides in this work are useful to examine fungal cell wall glycoprotein cross-linking by transglycosidase enzymes for antifungal drug development.

scholarly journals Cdc1 removes the ethanolamine phosphate of the first mannose of GPI anchors and thereby facilitates the integration of GPI proteins into the yeast cell wall

2014 ◽  
Vol 25 (21) ◽  
pp. 3375-3388 ◽  
Author(s):  
Hector M. Vazquez ◽  
Christine Vionnet ◽  
Carole Roubaty ◽  
Andreas Conzelmann
Keyword(s):  
Cell Wall ◽  
Secretory Pathway ◽  
Genetic Interaction ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Gpi Anchor ◽  
Distinct Cell ◽  
Gpi Proteins ◽  
Ethanolamine Phosphate

Temperature-sensitive cdc1tsmutants are reported to stop the cell cycle upon a shift to 30°C in early G2, that is, as small budded cells having completed DNA replication but unable to duplicate the spindle pole body. A recent report showed that PGAP5, a human homologue of CDC1, acts as a phosphodiesterase removing an ethanolamine phosphate (EtN-P) from mannose 2 of the glycosylphosphatidylinositol (GPI) anchor, thus permitting efficient endoplasmic reticulum (ER)-to-Golgi transport of GPI proteins. We find that the essential CDC1 gene can be deleted in mcd4∆ cells, which do not attach EtN-P to mannose 1 of the GPI anchor, suggesting that Cdc1 removes the EtN-P added by Mcd4. Cdc1-314tsmutants do not accumulate GPI proteins in the ER but have a partial secretion block later in the secretory pathway. Growth tests and the genetic interaction profile of cdc1-314tspinpoint a distinct cell wall defect. Osmotic support restores GPI protein secretion and actin polarization but not growth. Cell walls of cdc1-314tsmutants contain large amounts of GPI proteins that are easily released by β-glucanases and not attached to cell wall β1,6-glucans and that retain their original GPI anchor lipid. This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314tstransfer GPI proteins to cell wall β1,6-glucans inefficiently.

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