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P W Robbins

Publications and source records attributed to P W Robbins.

At least 19 recordsLinked to original sources
PubMed1992

Yeast glycoprotein biosynthesis: MNT1 encodes an alpha-1,2-mannosyltransferase involved in O-glycosylation.

The Saccharomyces cerevisiae MNT1 gene encodes a Golgi mannosyltransferase. Gene disruption of the MNT1 locus leads to a greater than 90% reduction of specific alpha-1,2-mannosyltransferase activity with alpha-methylmannoside as acceptor. Null mutants of MNT1 are viable, have no apparent growth defect, and are blocked in the elongation of protein O-linked mannobiose. Structural analysis of the N-linked outer chain isolated from an mnn1 mnn10 mnt1 strain revealed no alteration in carbohydrate structure compared to the parental mnn1 mnn10 strain. The MNT1 gene is identical to KRE2, and mutations in the gene render cells resistant to the killer toxin K1 of S. cerevisiae, which suggests a role for O-mannosylated proteins in the resistance mechanism. In addition, MNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases.

Carbohydrate Sequence↗
PubMed1992

Cloning and high-level expression of chitinase-encoding gene of Streptomyces plicatus.

A chitinase (Cht)-encoding gene from Streptomyces plicatus was previously cloned and expressed in Escherichia coli [Robbins et al., J. Biol. Chem., 263 (1988) 442-447]. We have sequenced this gene, compared its sequence with other genes encoding Cht and have explored its expression and regulation when reintroduced into Streptomyces lividans on multicopy plasmids. We have also cloned two other Streptomyces Cht-encoding genes and a beta-hexosaminidase-encoding gene in E. coli by expression in the lambda ZAP-Bluescript vector. The hexosaminidase and one of the Chts were expressed directly from the genomic library in E. coli at a high level as chimeric fusions with the beta-galactosidase alpha-complementing peptide encoded by the vector. Direct cloning and high-level expression of such chimeric proteins, which overcomes the difficulties associated with expressing Streptomyces genes in E. coli, should generally be possible wherever large numbers of transformants can be conveniently screened.

Amino Acid Sequence↗
PubMed1992

Classification of fungal chitin synthases.

Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (approximately 600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced amino acid sequences were aligned. With the exception of S. cerevisiae CHS1, the sequences fell into three distinct classes, which could represent separate functional groups. Within each class phylogenetic analysis was performed. Although not the major purpose of the investigation, this analysis tends to confirm some relationships consistent with current taxonomic groupings.

Amino Acid Sequence↗
PubMed1992

Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene.

A gene encoding an alpha-1,2-mannosyltransferase from Saccharomyces cerevisiae was cloned and sequenced. The alpha-1,2-mannosyltransferase which utilizes alpha-methylmannoside as acceptor of mannose from GDP-mannose was purified. The enzyme activity was shown to correspond to a 41 kDa protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This protein band was digested in situ with trypsin and amino acid sequence information was obtained from four peptides. Degenerate oligonucleotide primers corresponding to the amino acid sequences were designed and used for polymerase chain reactions on yeast genomic DNA. A specific reaction product was used to screen a genomic library of S.cerevisiae. A fragment of approximately 5.7 kb was isolated, of which a 2.9 kb fragment was sequenced. It contained a 1329 base pair open reading frame encoding the peptide sequences of the purified alpha-1,2-mannosyltransferase. The gene, designated MNT1, is located on the right arm of chromosome 4. It encodes a 442 amino acid polypeptide with a calculated mol. wt of 51.4 kDa. The corresponding mRNA has a length of approximately 1.6 kb. Overexpression of the MNT1 gene increased this alpha-1,2-mannosyltransferase activity approximately 2.5-fold. The protein was shown to be modified with N-linked carbohydrate chains and its sequence contains one N-glycosylation site. The enzyme contains a putative membrane-spanning domain near its N-terminus and its topology is thus similar to that of mammalian Golgi glycosyltransferases. This is the first report of the cloning and sequencing of a yeast Golgi mannosyltransferase.

Amino Acid Sequence↗
PubMed1992

Expression of chitin synthase genes during yeast and hyphal growth phases of Candida albicans.

Chitin, the beta 1,4-linked polymer of N-acetylglucosamine, is a fibrous polysaccharide that in many yeasts helps to maintain the structure of the mother-bud junction and in filamentous fungi is often the major supporting component of the cell wall. We have previously described a Candida albicans chitin synthase, CHS1. The DNA and derived protein sequences of a second gene, CHS2, are presented and compared with previously published gene sequences. Northern blot analysis shows that strikingly different levels of synthase 1 and 2 expression occur during yeast and hyphal phases of Candida growth.

Amino Acid Sequence↗
PubMed1991

Chitinase is required for cell separation during growth of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae chitinase described by Correa et al. (Correa, J. U., Elango, N., Polacheck, I., and Cabib, E. (1982) J. Biol. Chem. 257, 1392-1397) has been cloned and sequenced. Analysis of the derived amino acid sequence suggests that the protein contains four domains: a signal sequence, a catalytic domain, a serine/threonine-rich region, and a carboxyl-terminal domain with high binding affinity for chitin. Most of the enzyme produced by cells is secreted into the growth medium and is extensively glycosylated with a series of short O-linked mannose oligosaccharides ranging in size from Man2 to Man5. Chitinase O-mannosylation was further examined in the temperature-sensitive secretion mutants sec18, sec7, and sec6. Oligosaccharides isolated from chitinase accumulating in cells at the nonpermissive temperature revealed Man1 and Man2 associated with the sec18 mutant. sec6 and sec7 accumulated Man2-Man5 with a higher proportion of Man5 relative to the secreted protein. A significant amount of chitinase is also found associated with the cell wall through binding of COOH-terminal domain to chitin. Disruption of the gene for the enzyme leads to a defect in cell separation but does not substantially alter the level of cellular chitin.

Amino Acid Sequence↗
PubMed1991

Novel purification of the catalytic domain of Golgi alpha-mannosidase II. Characterization and comparison with the intact enzyme.

Rat liver alpha-mannosidase II, a hydrolase involved in the processing of asparagine-linked oligosaccharides, is an integral membrane glycoprotein facing the lumen of Golgi membranes. We have previously shown (Moremen, K. W., and Touster, O. (1986) J. Biol. Chem. 261, 10945-10951) that mild chymotrypsin digestion of permeabilized or solubilized Golgi membranes will result in the cleavage of the intact 124,000-dalton alpha-mannosidase II subunit, releasing a 110,000-dalton hydrophilic polypeptide which contains the catalytic site. Consistent with the removal of a membrane binding domain, the chymotrypsin-generated 110,000-dalton peptide was found exclusively in the aqueous phase in Triton X-114 phase separation studies, whereas the intact enzyme was found in the detergent phase. Taking advantage of this conversion in phase partitioning behavior, a purification procedure was developed to isolate the 110,000-dalton proteolytic digestion product as a homogeneous polypeptide for further characterization and protein sequencing at a yield of greater than 65% from a rat liver Golgi-enriched membrane fraction. An improved purification procedure for the intact enzyme was also developed. The two forms of the enzyme were compared yielding the following results. (a) The catalytic activity of the intact and cleaved forms of alpha-mannosidase II were indistinguishable in Km, Vmax, inhibition by the alkaloid, swainsonine, and in their activity toward the natural substrate GlcNAc-Man5GlcNAc. (b) Both the intact and cleaved forms of the enzyme appear to be disulfide-linked dimers. (c) The two forms of the enzyme contain different NH2-terminal sequences suggesting that the cleaved NH2 terminus contains the membrane-spanning domain. (d) Additional peptide sequences were obtained from proteolytic fragments and cyanogen bromide digestion products in order to create a partial protein sequence map of the enzyme. These results are consistent with a model common among Golgi processing enzymes of a hydrophilic catalytic domain anchored to the lumenal face of Golgi membranes through an NH2-terminal hydrophobic membrane-anchoring domain.

Amino Acid Sequence↗
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↗
PubMed1991

Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans.

Golgi alpha-mannosidase II (GlcNAc transferase I-dependent alpha 1,3[alpha 1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures. We have isolated overlapping clones from a murine cDNA library encoding the full length alpha-mannosidase II open reading frame and most of the 5' and 3' untranslated region. The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids). This domain organization which is shared with the Golgi glycosyl-transferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization. Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as much as 2,543 bp. Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message. The largest and predominant message on Northern blots (7.5 kb) presumably extends another approximately 1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the alpha-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of cross-reactive material in a perinuclear membrane array consistent with a Golgi localization. A region within the catalytic domain of the alpha-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum alpha-mannosidase and the vacuolar alpha-mannosidase of Saccharomyces cerevisiae. Partial human alpha-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5.

Amino Acid Sequence↗
PubMed1990

A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization.

We have recently described a luminal guanosine diphosphatase activity in Golgi-like vesicles of Saccharomyces cerevisiae (Abeijon, C., Orlean, P., Robbins, P. W., and Hirschberg, C. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6935-6939). The presumed in vivo role of this enzyme is to convert GDP into GMP. GDP is a reaction product following outer-chain mannosylation of luminal proteins and a known inhibitor of mannosyltransferases. It is hypothesized that GMP then returns to the cytosol. We have purified this enzyme to apparent homogeneity. Following solubilization from a membrane pellet using a buffer containing Triton X-100, the enzyme was purified on a concanavalin A-Sepharose column followed by Mono Q fast protein liquid chromatography (FPLC) and Superose-12 FPLC columns. After treatment with endoglycosidase H, the deglycosylated active enzyme was applied to a second Mono Q FPLC column and a phenyl-Superose FPLC column. The final enzyme activity was enriched 6500-fold over that of the Triton X-100 extract. The apparant molecular mass of the deglycosylated enzyme is 47 kDa. The purified enzyme is highly specific for guanosine diphosphate, requires Ca2+ for maximal activity, and has a broad pH optimum between 7.4 and 8.2. The apparent Km for GDP is 0.1 mM; the Vmax is 4.9 mmol/min/mg of protein. An enzyme activity with similar substrate specificity has also been detected in membranes of Schizosaccharomyces pombe.

Chromatography, Affinity↗
PubMed1990

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae.

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).

Amino Acid Sequence↗
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

Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man2GlcNAc2 and Man1GlcNAc2 are transferred from dolichol to protein in vivo.

Transfer of truncated oligosaccharides to protein in vivo and the structure of Man2GlcNAc2 synthesized by intact yeast (Saccharomyces cerevisiae) were investigated in the alg2 mutant. At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man2GlcNAc2 and Man1GlcNAc2 (T.C. Huffaker and P.W. Robbins (1983) Proc. Natl. Acad. Sci. USA 80, 7466-7470). We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC. Man2GlcNAc2 and Man1GlcNAc2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) dependent. Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype. The alg2/pAC3 cells are viable at 36 degrees C. Two isomers of Man2GlcNAc2, Man1----3ManGlcNAc2 and Man1----6ManGlcNAc2, were present on lipid and protein. The transfer of Man2GlcNAc2 and Man1GlcNAc2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.

Borohydrides↗
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↗