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.     

The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.     

Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.     

Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation

In budding yeast, at least 10 proteins are required for formation of the double-strand breaks (DSBs) that initiate meiotic recombination. Spo11 is the enzyme responsible for cleaving DNA and is found in a complex that also contains Ski8, Rec102, and Rec104. The Mre11/Rad50/Xrs2 complex is required for both DSB formation and DSB processing. In this article we investigate the functions of the remaining three proteins--Mer2, Mei4, and Rec114--with particular emphasis on Mer2. The Mer2 protein is present in vegetative cells, but it increases in abundance and becomes phosphorylated specifically during meiotic prophase. Mer2 localizes to distinct foci on meiotic chromosomes, with foci maximally abundant prior to the formation of synaptonemal complex. If DSB formation is blocked (e.g., by a spo11 mutation), dephosphorylation of Mer2 and its dissociation from chromosomes are delayed. We have also found that the Mei4 and Rec114 proteins localize to foci on chromosomes and these foci partially colocalize with each other and with Mer2. Furthermore, the three proteins co-immunoprecipitate. Mer2 does not show significant colocalization with Mre11 or Rec102 and Mer2 does not co-immunoprecipitate with Rec102. We propose that Mer2, Mei4, and Rec114 form a distinct complex required for DSB formation.     

Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase

The formation of N-glycosidic linkages of glycoproteins involves the ordered assembly of the common Glc3Man9GlcNAc2 core-oligosaccharide on the lipid carrier dolichyl pyrophosphate. Whereas early mannosylation steps occur on the cytoplasmic side of the endoplasmic reticulum with GDP-Man as donor, the final reactions from Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol on the lumenal side use Dol-P-Man. We have investigated these later stages in vitro using a detergent-solubilized enzyme extract from yeast membranes. Mannosyltransfer from Dol-P-Man to [3H]Man5GlcNAc2-PP-Dol with formation of all intermediates up to Man9GlcNAc2-PP-Dol occured in a rapid, time- and protein-dependent fashion. We find that the initial reaction from Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol is independent of metal ions, but further elongations need Mn2+ that can be partly replaced by Mg2+ or Ca2+. Zn2+ or Cd2+ ions were found to inhibit formation of Man(7-9)GlcNAc2-PP-Dol, but do not affect synthesis of Man6GlcNAc2-PP-Dol. Extension did not occur when the acceptor was added as a free Man5GlcNAc2 oligosaccharide or when GDP-Man was used as mannosyl donor. The alg3 mutant was described to accumulate Man5GlcNAc2-PP-Dol. We expressed a functional active HA-epitope tagged ALG3 fusion and succeeded to selectively immunoprecipitate the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase activity from the other enzymes of the detergent extract involved in the subsequent mannosylation reactions. This demonstrates that Alg3p represents the mannosyltransferase itself and not an accessory protein involved in the reaction.     

Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase

The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose was attached to the 6-linked mannose in an alpha 1,3-linkage. This linkage was further confirmed by acetolysis of the oligosaccharide product [i.e., Man6(GlcNAc)2], which gave a labeled disaccharide as the major product (greater than 90%).     

Interaction between the C termini of Alg13 and Alg14 mediates formation of the active UDP-N-acetylglucosamine transferase complex

The second step of eukaryotic N-linked glycosylation in endoplasmic reticulum is catalyzed by an UDP-N-acetylglucosamine transferase that is comprised of two subunits, Alg13 and Alg14. The interaction between Alg13 and 14 is crucial for UDP-GlcNAc transferase activity, so formation of the Alg13/14 complex is likely to play a key role in the regulation of N-glycosylation. Using a combination of bioinformatics and molecular biological methods, we have undertaken a functional analysis of yeast Alg13 and Alg14 proteins to elucidate the mechanism of their interaction. Our mutational studies demonstrated that a short C-terminal alpha-helix of Alg13 is required for interaction with Alg14 and for enzyme activity. Electrostatic surface views of the modeled Alg13/14 complex suggest the presence of a hydrophobic cleft in Alg14 that provides a pocket for the Alg13 C-terminal alpha-helix. Co-immunoprecipitation assays confirmed the C-terminal three amino acids of Alg14 are required for maintaining the integrity of Alg13/Alg14 complex, and this depends on their hydrophobicity. Modeling studies place these three Alg14 residues at the entrance of the hydrophobic-binding pocket, suggesting their role in the stabilization of the interaction between the C termini of Alg13 and Alg14. Together, these results demonstrate that formation of this hetero-oligomeric complex is mediated by a short C-terminal alpha-helix of Alg13 in cooperation with the last three amino acids of Alg14. In addition, deletion of the N-terminal beta-strand of Alg13 caused the destruction of protein, indicating the structural importance of this region in protein stability.     

Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation

N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc2-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc2-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor.     

Biosynthesis of lipid-linked oligosaccharides. Isolation and structure of a second lipid-linked oligosaccharide in Chinese hamster ovary cells.

Previous work has shown that vesicular stomatitis virus-infected Chinese hamster ovary cells contain a major high molecular weight lipid-linked oligosaccharide which is transferred en bloc to protein during the formation of the asparagine-linked complex-type oligosaccharides of the vesicular stomatitis virus G protein (Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722). We now report the characterization of a second, lower molecular weight lipid-linked oligosaccharide. The oligosaccharide portion of this molecule was isolated and its structure was determined by methylation analysis, digestion with exoglycosidases, acetolysis and Smith periodate degradation to be: (formula: see text). Several lines of evidence are presented which indicate that this lipid-linked oligosaccharide is primarily involved in the assembly of the major lipid-linked oligosaccharide rather than in the direct glycosylation of proteins.     

The ubiquitin ligase SCF(Grr1) is required for Gal2p degradation in the yeast Saccharomyces cerevisiae

F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCF(Grr1), including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCF(Grr1)-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1Delta mutant cells imply that Gal2p is not the primary target of SCF(Grr1) ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.     

Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p

Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length. It is documented that Cdc13p binds specifically to single-stranded TG_1–3 telomeric DNA sequences and interacts with Stn1p. To localize the region for single-stranded TG_1–3 DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity. Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451–693) was sufficient to bind single-stranded TG_1–3 with specificity similar to that of the native protein. Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres. However, the telomere-binding region of Cdc13p (amino acids 451–693) was not capable of complementing the growth defects of cdc13 mutants. Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252–924) complemented the growth defects of cdc13 mutants. These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required. Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.     

Functional domains required for the Saccharomyces cerevisiae Mus81-Mms4 endonuclease complex formation and nuclear localization

The Saccharomyces cerevisiae Mms4 and Mus81 proteins form a specific complex, which functions as an endonuclease specific for branched DNA molecules and protects cells from killing by DNA alkylation damage, but not damage induced by ionizing radiations. In an effort to further understand the structure and functions of the Mus81-Mms4 complex, we attempted to define domains required for complex formation and nuclear localization through deletion and mutagenesis analyses. Combined yeast two-hybrid and co-immunoprecipitation experiments indicate that the C-terminal 100 amino acids of both Mus81 and Mms4 are required and sufficient for heterodimer formation. However, a single amino acid substitution in Mms4 in the N-terminal region is able to abolish the interaction, which suggests that the three-dimensional structure is also important for Mms4 to interact with Mus81. By fusion to green fluorescent protein and in vivo subcellular localization studies, we demonstrate that Mms4 and Mus81 are nuclear proteins and can be localized to the nucleus independently. Deletion analyses indicate that one of two putative nuclear localization signals (residues 244-263) in Mms4 is required for localization, whereas the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.     

The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the products of at least 15 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Previously, we have shown that three of these genes, SEC6, SEC8 and SEC15, encode components of a multisubunit complex which localizes to the tip of the bud, the predominant site of exocytosis in S. cerevisiae. Mutations in three more of these genes, SEC3, SEC5 and SEC10, were found to disrupt the subunit integrity of the Sec6-Sec8-Sec15 complex, indicating that these genes may encode some of the remaining components of this complex. To examine this possibility, we cloned and sequenced the SEC5 and SEC10 genes, disrupted them, and either epitope tagged them (Sec5p) or prepared polyclonal antisera (Sec10p) to them for co-immunoprecipitation studies. Concurrently, we biochemically purified the remaining unidentified polypeptides of the Sec6-Sec8-Sec15 complex for peptide microsequencing. The genes encoding these components were identified by comparison of predicted amino acid sequences with those obtained from peptide microsequencing of the purified complex components. In addition to Sec6p, Sec8p and Sec15p, the complex contains the proteins encoded by SEC3, SEC5, SEC10 and a novel gene, EXO70. Since these seven proteins function together in a complex required for exocytosis, and not other intracellular trafficking steps, we have named it the Exocyst.     

Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in culture

The lipid-linked oligosaccharides synthesized in the presence of the alpha-glucosidase inhibitors, 1-deoxynojirimycin (DJN) and N-methyl-1-deoxynojirimycin (MDJN), were compared in IEC-6 intestinal epithelial cells in culture. HPLC analysis of the oligosaccharides obtained before and after exhaustive jack bean alpha-mannosidase digestion indicates that control and MDJN-treated cells synthesize similar amounts of Glc3Man9GlcNAc2-PP-dolichol. In contrast, the formation of this compound is greatly reduced in DJN-treated cells, the major lipid-linked oligosaccharide found being Man9GlcNAc2-PP-dolichol. The decreased availability of the preferred donor for protein glycosylation may account for the impaired glycosylation and secretion of certain glycoproteins in the presence of DJN.     

Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3'' end formation of 5.8S rRNA in Saccharomyces cerevisiae.

The temperature-sensitive mutation, dob1-1, was identified in a screen for dependence on overexpression of the yeast translation initiation factor eIF4B (Tif3p). Dob1p is an essential putative ATP-dependent RNA helicase. Polysome analyses revealed an under accumulation of 60S ribosomal subunits in the dob1-1 mutant. Pulse-chase labelling of pre-rRNA showed that this was due to a defect in the synthesis of the 5.8S and 25S rRNAs. Northern and primer extension analyses in the dob1-1 mutant, or in a strain genetically depleted of Dob1p, revealed a specific inhibition of the 3' processing of the 5.8S rRNA from its 7S precursor. This processing recently has been attributed to the activity of the exosome, a complex of 3'-->5' exonucleases that includes Rrp4p. In vivo depletion of Dob1p also inhibits degradation of the 5' external transcribed spacer region of the pre-rRNA. A similar phenotype was observed in rrp4 mutant strains and, moreover, the dob1-1 and rrp4-1 mutations show a strong synergistic growth inhibition. We propose that Dob1p functions as a cofactor for the exosome complex that unwinds secondary structures in the pre-rRNA that otherwise block the progression of the 3'-->5' exonucleases.     

Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae

The Saccharomyces cerevisiae MNN4 gene, which is involved inmannosylphosphate transfer from GDP-mannose to N-linked oligosaccharide,has been cloned from a lambda phage containing a yeast chromosomeXI DNA fragment The MNN4 ORF encodes a protein of 1178 aminoacids. The deduced amino acid sequence shows a topology of typeII membrane proteins and has a unique repeated sequence of lysineand glutamic acid at the C-terminus. Disruption and overexpressionof MNN4 led to a decrease and increase, respectively, of themannosylphosphate content in cell wall mannans prepared fromboth mnn4 and wild type strains. A dramatic decrease of mannosylphosphateoccurs in     

Crystal structure of the oxygen-dependant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family. Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 A. The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded anti-parallel sheet that is flanked by helices. The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a beta-ladder with its counterpart in the partner subunit. The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms. A substratesized cavity is completely buried in the closed conformation by the approximately 8-A movement of a helix that forms a lid over the active site. The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.     

Aux1p/Swa2p is required for cortical endoplasmic reticulum inheritance in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus. The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event. To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p). This approach has revealed that AUX1/SWA2 plays a role in ER inheritance. The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles. Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells. These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance. In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.