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P Orlean

Publications and source records attributed to P Orlean.

At least 37 records · Page 2Linked to original sources
PubMed1993

Glycoprotein biosynthesis in yeast.

Many proteins in the yeast Saccharomyces cerevisiae are modified by the attachment of N-linked saccharides to asparagine, of O-linked mannose glycans to serine or threonine, and of glycosylphosphoinositol membrane anchors. The biosynthetic events leading to these modifications are coupled to the secretory pathway. Early stages of N-linked glycosylation and the formation of glycosylphosphoinositol anchors have been conserved through evolution of eukaryotes. Studies of yeast offer a variety of genetic and molecular biological approaches, which have led to the isolation of different glycosylation mutants and of genes for enzymes involved in glycosylation. Yeast mutants are useful to identify biosynthetic intermediates, to establish whether a given enzyme is essential for viability, and to determine how cellular functions are affected when glycosylation is perturbed. Yeast glycosylation mutants and genes can be used to identify their counterparts in other eukaryotes.

Carbohydrate Metabolism↗
PubMed1992

Saccharomyces cerevisiae sec59 cells are deficient in dolichol kinase activity.

The temperature-sensitive Saccharomyces cerevisiae mutant sec59 accumulates inactive and incompletely glycosylated protein precursors in its endoplasmic reticulum at the restrictive temperature. O-mannosylation and glycosyl phosphatidylinositol membrane anchoring of protein are also abolished, consistent with a deficiency in dolichyl phosphate mannose. Membranes prepared from sec59 cells that had been shifted to the restrictive temperature, however, made normal amounts of dolichyl phosphate mannose when exogenous dolichyl phosphate was supplied, but dolichyl phosphate mannose synthesis was severely depressed in the absence of exogenous dolichyl phosphate. Quantitative measurements of dolichyl phosphate in sec59 cells showed that the levels were decreased to 48% of wild type at the permissive temperature and to less than 10% at the restrictive temperature. Assays of enzymes from the dolichyl phosphate synthetic pathway, cis-prenyltransferase and dolichyl pyrophosphate phosphatase, gave wild-type levels. However, dolichol kinase activity was greatly decreased. When sec59 cells were transformed with a plasmid that overexpresses the wild-type gene, dolichol kinase activity increased 10-fold over wild-type levels. These results strongly suggest that the sec59 gene encodes dolichol kinase.

Dimethylallyltranstransferase↗
PubMed1992

Inositol acylation of a potential glycosyl phosphoinositol anchor precursor from yeast requires acyl coenzyme A.

Glycosyl phosphoinositol (GPI) anchors on proteins can be modified by palmitoylation of their inositol residue, which makes such anchors resistant to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) (Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T.L. (1988) J. Biol. Chem. 263, 18766-18775). Mannosylated GPI lipids made in trypanosomal and mammalian cells can also be inositol-acylated, indicating that inositol acylation may be a normal step in GPI anchor synthesis. We find that Saccharomyces cerevisiae mutants blocked in dolichyl phosphate mannose synthesis accumulate a lipid that can be radiolabeled in vivo with [3H]myo-inositol, [3H]GlcN, and [3H]palmitic acid. This lipid is resistant to PI-PLC, yet sensitive to mild alkaline hydrolysis, and has been characterized as GlcN-phosphatidylinositol (PI), fatty acylated on its inositol residue. When yeast membranes are incubated with UDP-[14C] GlcNAc, 14C-labeled GlcNAc-PI and GlcN-PI are made. Addition of ATP and CoA, or of palmitoyl-CoA to incubations results in the synthesis of [14C]GlcN-(acyl-inositol)PI. This lipid is also made when membranes are incubated with [1-14C]palmitoyl-CoA and UDP-GlcNAc. We propose that acyl CoA is the donor in inositol acylation of GlcN-PI, and that GlcN-(acyl-inositol)PI is an obligatory intermediate in GPI synthesis.

Acyl Coenzyme A↗
PubMed1992

Enzymes that recognize dolichols participate in three glycosylation pathways and are required for protein secretion.

We have explored the structure, function, and membrane topography of enzymes that recognize dolichols and participate in glycosylation pathways in the endoplasmic reticulum. Enzymes that interact with dolichols, including dolichyl phosphate mannose (Dol-P-Man) synthase and UDP-GlcNAc:Dol-P-transferase, revealed a conserved amino acid sequence in membrane-spanning regions. The consensus is Phe-Ile/Val-Xaa-Phe/Try-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe/Tyr, and we propose it is involved in dolichol recognition. We have used yeast mutants to demonstrate the role of dolichols in three glycosylation pathways. At its nonpermissive temperature, a Dol-P-Man synthase mutant (dpm1) was blocked in N-glycosylation, O-mannosylation, and glycosyl phosphoinositol membrane anchoring of protein, most likely because Dol-P-Man serves as mannosyl donor in all three pathways. The secretion mutant sec59 has a similar phenotype to dpm1, and the presence of a dolichol recognition sequence in the SEC59 protein gave a clue to its defect, which is in dolichol kinase. Comparison of yeast glycosylation mutant suggests that the ability to carry out N-glycosylation alone is sufficient to allow yeast to secrete glycoproteins and that an N-linked saccharide of a minimum size must be attached to proteins for cells to be able to secrete them and maintain a functional secretory pathway.

Amino Acid Sequence↗
PubMed1991

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.

Antigens, CD↗
PubMed1991

Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy.

The endoplasmic reticulum (ER) and other secretory compartments of Saccharomyces cerevisiae have biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct. In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content. These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of the S. cerevisiae ER structure, both in intact cells and at the ultrastructural level. Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane. Double-labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment. This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycles.

Cell Cycle↗
PubMed1990

Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.

Animals↗
PubMed1990

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

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.

Carbohydrate Conformation↗
PubMed1990

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.

Animals↗
PubMed1989

Topography of glycosylation in yeast: characterization of GDPmannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles.

"Outer-chain" addition of mannose residues to yeast glycoproteins occurs in the Golgi compartment of the cell. Essential steps in this process are thought to include transport of GDPmannose from the cytoplasm into the lumen of Golgi vesicles, transfer of mannose to glycoprotein acceptors, hydrolysis of the resulting GDP to GMP, and return of GMP and inorganic phosphate to the cytoplasm. We report detection and characterization of a GDPmannose transport activity and a GDPase by yeast vesicles. The active transport of GDPmannose as well as the GDPase and another presumed Golgi enzyme, alpha 1,2-mannosyltransferase, are concentrated in a subcellular fraction that can be partially separated, by velocity sucrose gradient centrifugation, from a fraction enriched in an endoplasmic reticulum marker enzyme.

Biological Transport↗
PubMed1989

A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition.

A 13-amino acid peptide was identified in three glycosyltransferases of the yeast endoplasmic reticulum. These enzymes, the products of the ALG1, ALG7, and DPM1 genes, catalyze the transfer of sugars from nucleotide sugars to dolichol phosphate derivatives. The consensus sequence for the conserved peptide was Leu-Phe-Val-Xaa-Phe-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe-Tyr. A sequence resembling the conserved peptide was also found in the predicted SEC59 protein, which is suspected to participate in assembly of the lipid-linked precursor oligosaccharide, although its specific function is unknown. All of the identified sequences contain an isoleucine at position 8 and phenylalanine or tyrosine at positions 2, 5, and 12. We believe this peptide may be involved in dolichol recognition for the following reasons. (i) The conserved sequence occurs in potential membrane-spanning regions. (ii) The ALG7 and DPM1 proteins are known to recognize the isoprenoid region of dolichol phosphate specifically; this recognition presumably occurs in the membrane since dolichol is very hydrophobic. (iii) The consensus sequence is similar to a region of two halobacterial proteins implicated in binding of the isoprenoid region of retinal. (iv) If the consensus sequence is represented as an alpha-helix, the conserved residues lie on one face of the helix. An alpha-helical structure is likely since the conserved regions are in potential membrane-spanning domains.

Amino Acid Sequence↗
PubMed1988

Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein.

Dolichol phosphate mannose (Dol-P-Man) synthase (EC 2.4.1.83) catalyzes the formation of Dol-P-Man from Dol-P and GDP-Man. The structural gene for yeast Dol-P-Man synthase (DPM1) was isolated by screening a yeast genomic DNA library for colonies that overexpressed Dol-P-Man synthase activity. This approach relied on a method to screen for Dol-P-Man synthase activity in lysed yeast colonies and used a yeast mutant with very low Dol-P-Man synthase activity in colony lysates. Transformants isolated using this technique expressed Dol-P-Man synthase activity 9-14-fold higher than that of a wild type strain, and all seven plasmids conferring this overproduction had a common region in their yeast genomic DNA insert. DPM1 is the structural gene for yeast Dol-P-Man synthase since Escherichia coli transformants harboring this gene express Dol-P-Man synthase activity in vitro. DNA sequencing of the DPM1 gene revealed an open reading frame of 801 bases. The 30-kDa size of the predicted protein is in excellent agreement with the size of the purified yeast enzyme (Haselbeck, A., and Tanner, W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1520-1524). Analysis of the predicted amino acid sequence reveals the protein has a potential membrane spanning domain of 25 amino acids at its COOH terminus. The protein's NH2 terminus, though not hydrophobic, meets existing criteria for yeast signal sequences, but there is no site for cleavage by signal peptidase. If the NH2 terminus is a functional signal sequence, the protein is predicted to be oriented toward the lumen of the endoplasmic reticulum with both NH2 and COOH termini serving as membrane anchors. If there is no signal sequence, the enzyme is predicted to face the cytoplasm and be anchored only by its COOH terminus. The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal. We suspect Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth. Instead, DPM1 may be an essential gene because its product is required for O-glycosylation in yeast or because Dol-P-Man synthase is needed in some unidentified pathway.

Alleles↗
PubMed1987

Two chitin synthases in Saccharomyces cerevisiae.

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.

Cations, Divalent↗
PubMed1986

Synthesis of an O-glycosylated cell surface protein induced in yeast by alpha factor.

A number of cell surface glycoproteins can be specifically and completely released from intact cells of Saccharomyces cerevisiae with 0.5% mercaptoethanol. Among these proteins is one with a molecular mass of 22 kDa, which is synthesized only in haploid a cells treated with the peptide mating pheromone alpha factor. This protein could be radiolabeled in vivo with [2-(3)H]mannose, [(14)C]phenylalanine, and [(35)S]sulfate. Its synthesis and export to the cell surface were not inhibited by tunicamycin. beta-Elimination released almost all radioactivity from the [2-(3)H]mannose-labeled protein, 36% of its radioactivity being recovered subsequently as mannose and 43% as a dimannoside. Evidence is presented that the 22-kDa O-glycosylated protein is a mating-type specific a cell agglutinin.

Journal Article↗
PubMed1985

Apparent inhibition of glycoprotein synthesis by S.cerevisiae mating pheromones.

Saccharomyces cerevisiae mating pheromones a and alpha factor strongly inhibit the incorporation of radiolabelled glucosamine into N-glycosylated proteins of corresponding haploid cells. This observation was erroneously interpreted as an inhibition of glycoprotein synthesis. It has turned out that alpha factor causes a 4-5-fold dilution of incorporated [14C]glucosamine with non-radioactive endogenous precursor. In the case of the [14C]chitin synthesized, which does not show inhibition by alpha factor, the lowering of the specific activity of the precursor is exactly compensated for by an increased rate of chitin synthesis caused by alpha factor.

Animals↗
PubMed1984

Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N-glycosylated.

alpha Factor specifically inhibits the synthesis of N-glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in alpha cells or in a/alpha diploids. a Factor has the same effect of alpha cells. The synthesis of O-glycosylated proteins is not inhibited. Although the mating pheromones act like a 'physiological tunicamycin', the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N-glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in alpha-factor-treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by alpha factor; but only in the former case did carbohydrate-free protein accumulate in the cells. alpha Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N-glycosylated proteins are required for the G1/S-phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature-sensitive N-glycosylation mutant is shifted to its restrictive temperature.

Animals↗
PubMed1984

Glycogen synthesis by cell-free extracts from the dimorphic yeast Candida albicans.

A particulate glucosyltransferase prepared from budding and filamentous cultures of Candida albicans used uridine diphosphate glucose as sole glucosyl donor in a reaction (measured by following the incorporation of [14C]-glucose from UDP [14C]-glucose into polymer) stimulated by glucose-6-phosphate and inhibited by adenosine triphosphate and guanosine triphosphate. The radiolabelled reaction product was solubilized by alpha-amylase, and, on oxidation with periodate followed by reduction with borohydride and acid hydrolysis, yielded erythritol and glycerol in the ratio of 4 to 1. The radiolabelled glucosyl residues were attached to an endogenous acceptor of high molecular weight.

Adenosine Triphosphate↗