Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1990
The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.
The Journal of biological chemistry, 1994
The protein gp115 is an exocellular yeast glycoprotein modified by O- and N-glycosylation and attached to the plasma membrane through a glycosylphosphatidylinositol. The more remarkable structural feature in gp115 is the presence of a 36-amino acid serine-rich region. Similar sequences have been found in mammalian glycoproteins, such as the low density lipoprotein receptor, the decay-accelerating factor, and the mucins, where they are targets of multiple sites of O-glycosylation. The modification of these regions greatly influences their conformation and gives rise to "rodlike" structures. In this work, we have deleted or duplicated the Ser-rich region of gp115. The analysis of the size and glycosylation state of both mutant proteins indicates that about 52% of the total contribution of the O-glycosylation to the mass of the protein is concentrated in this region. The phenotype of ggp1 null mutant expressing the mutant proteins was also analyzed to understand if this regio...
Journal of bacteriology, 1993
This paper reports a phenotypic characterization of ggp1 mutants. The cloned GGP1 (GAS1) gene, which encodes a major GPI-anchored glycoprotein (gp115) of Saccharomyces cerevisiae of unknown function, was used to direct the inactivation of the chromosomal gene in haploid and diploid strains by gene replacement. The analysis of the null mutants reveals a reduction in the growth rate of 15 to 40%. Cells are round, with more than one bud, and extensively vacuolized. In the stationary phase, mutant cells are very large, arrest with a high percentage of budded cells (about 54 and 70% for haploid and diploid null mutants, respectively, in comparison with about 10 to 13% for control cells), and have reduced viability. The observed phenotype suggests defects in cell separation. Flow cytometric analysis of DNA reveals an increase in the fraction of cells in the G2+M+G1* compartment during exponential growth. Conjugation and sporulation are not affected. The exocellular location of gp115 led u...
Crosstalk between protein N-glycosylation and lipid metabolism in Saccharomyces cerevisiae
Scientific Reports, 2019
the endoplasmic reticulum (eR) is a multi functional organelle and plays a crucial role in protein folding and lipid biosynthesis. the SEC59 gene encodes dolichol kinase, required for protein glycosylation in the eR. the mutation of sec59-1 caused a protein n-glycosylation defect mediated eR stress resulting in increased levels of phospholipid, neutral lipid and sterol, whereas growth was reduced. in the sec59-1∆ cell, the N-glycosylation of vacuolar carboxy peptidase-Y (CPY) was significantly reduced; whereas the ER stress marker Kar2p and unfolded protein response (UPR) were significantly increased. Increased levels of triacylglycerol (tAG), sterol ester (Se), and lipid droplets (LD) could be attributed to upregulation of DPP1, LRO1, and ARE2 in the sec 59-1∆ cell. Also, the diacylglycerol (DAG), sterol (Ste), and free fatty acids (FFA) levels were significantly increased, whereas the genes involved in peroxisome biogenesis and Pex3-EGFP levels were reduced when compared to the wild-type. The microarray data also revealed increased expression of genes involved in phospholipid, tAG, fatty acid, sterol synthesis, and phospholipid transport resulting in dysregulation of lipid homeostasis in the sec59-1∆ cell. We conclude that SEC59 dependent n-glycosylation is required for lipid homeostasis, peroxisome biogenesis, and eR protein quality control. Proteins synthesized in the ER are post-translationally modified by N-glycosylation and O-glycosylation. The glycosylation process starts in the ER, and extends to the Golgi apparatus, which requires activated dolichol precursors for the initial steps. The vast majorities of secretory proteins are N-linked glycoproteins and play an important role in protein secretion, morphogenesis, and development of multi-cellular organisms. SEC59 (SECretory) encodes dolichol kinase (DK) and catalyzes CTP-dependent phosphorylation of dolichol. Dolichol kinase transfers the phosphoryl group from CTP to dolichol and catalyzes the final step of the de novo pathway for the Dol-P formation. CTP-mediated dolichol kinase is involved in recycling of glycosyl carrier lipid after it is discharged as Dol-P-P in N-glycosylation reactions. On the ER luminal surface the Dol-P-P phosphatase converts Dol-P-P to Dol-P, and is diffused back to the cytoplasmic leaflet of the ER. Dol-P (Dolichol Monophosphate) serves as a glycosyl carrier lipid in the assembly of N-linked glycoproteins, glycosylphosphatidylinositol anchors, and C and O-mannosylation. The DK is involved in the dolichol monophosphate (Dol-P) synthesis and serves as a carrier lipid in the assembly of N-linked glycoproteins 1. When the accumulation of unfolded or misfolded proteins in the ER activates the transmembrane kinase/nuclease Ire1p 2 , and initiates the HAC1 mRNA splicing. The Hac1p, a βZIP transcription factor induced the unfolded protein response (UPR) target genes 2. The activation of the UPR allows the cell to tolerate stress and presumably assist in a correction of the insult that is caused by unfolded protein accumulation 2. During the ER stress, the misfolded or unfolded proteins are accumulated in the ER, further transported to the cytosol, and eliminated by the proteasomal degradation 3. When there is a defect in protein folding, the ER-associated degradation (ERAD) is enhanced. The UPR and ERAD pathways are responsible for removal of the aberrant proteins from the ER 3. The protein glycosylation and protein quality control homeostasis are crucial and evolutionarily conserved from yeast to human 4. The N-glycosylation defects are
Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae
Molecular and cellular biology, 1989
In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed. GPH1 was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid. Northern (RNA) analy...
Yeast, 1996
The GGPlIGASIICWH52 gene of Succhuronzyce.r cerevisiue encodes a major exocellular 1 15 kDa glycoprotein (gpll5) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gpll5 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. P H R l gene isolated from Ctindidu alibicons is homologous to the GGPl gene. In this report we have analysed the ability of P H R l to complement a ggplA mutation in S. cerevisiue. The expression of P H R l controlled by its natural promoter or by the GGPl promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that P H R l in budding yeast gives rise to a 75-80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. ull,icuns gene product is functional in S. cerevisirce.
Journal of Biological Chemistry, 1999
Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in ⌬gpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9 -11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiesterlinked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the ␣1,6-linked mannose of the GPI core structure.