Ludwig Lehle

Protein glycosylation in yeast

S. cerevisiae contains many mannose-rich glycoproteins that possess N- and O-linked carbohydrate chains, and both types may even occur on one and the same protein. The steps in the synthesis of asparagine-linked chains begin with assembly and transfer of the lipid-linked precursor to protein in a way common to all eucaryotes. Subsequent modifications lead to mannosyl extensions of various lengths, but complex type carbohydrate structures are not formed. Oligosaccharides O-linked to serine/threonine consist exclusively of mannose in S. cerevisiae. The mannose residue attached directly to the protein is transferred from Dol-P-Man in a unique way, which has been observed so far for fungal cells only. The cellular localization of the glycosylation reactions is summarized and the problem of transmembrane translocation of the sugar precursors at the ER and the Golgi is discussed. Some aspects of secretory (sec) and asparagine linked glycosylation (alg) mutants have been covered, and the various hypotheses related to the possible functions of this costly protein modification process are discussed. The article may also be helpful for those, who want to exploit the yeasts protein synthesizing machinery by genetically manipulating the cells.

The oligosaccharyltransferase complex from yeast

N-Glycosylation of eukaryotic secretory and membrane-bound proteins is an essential and highly conserved protein modification. The key step of this pathway is the en bloc transfer of the high mannose core oligosaccharide Glc3Man9GlcNAc2 from the lipid carrier dolichyl phosphate to selected Asn-X-Ser/Thr sequences of nascent polypeptide chains during their translocation across the endoplasmic reticulum membrane. The reaction is catalysed by the enzyme oligosaccharyltransferase (OST). Recent biochemical and molecular genetic studies in yeast have yielded novel insights into this enzyme with multiple tasks. Nine proteins have been shown to be OST components. These are assembled into a heterooligomeric membrane-bound complex and are required for optimal expression of OST activity in vivo in wild type cells. In accord with the evolutionary conservation of core N-glycosylation, there are significant homologies between the protein sequences of OST subunits from yeast and higher eukaryotes, and OST complexes from different sources show a similar organisation as well.

STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo

N‐linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid‐linked core‐oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid‐linked core‐oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N‐linked glycosylation. Besides novel mutants deficient in the assembly of the lipid‐linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3–3 cells very little glycosyl transfer occurs from incomplete lipid‐linked oligosaccharide, whereas the transfer of full‐length Glc3Man9GlcNAc2 is hardly affected as compared with wild‐type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.

SRD5A3 Is Required for Converting Polyprenol to Dolichol and Is Mutated in a Congenital Glycosylation Disorder

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies

Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.

Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the γ-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of β-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro.

Asparagine‐linked N‐glycosylation is a highly conserved and functionally important modification of proteins in eukaryotic cells. The central step in this process is a cotranslational transfer of lipid‐linked core oligosaccharides to selected Asn‐X‐Ser/Thr‐sequences of nascent polypeptide chains, catalysed by the enzyme N‐oligosaccharyl transferase. In this report we show that the essential yeast protein WBP1 (te Heesen et al., 1991) is required for N‐oligosaccharyl transferase in vivo and in vitro. Depletion of WBP1 correlates with a defect in transferring core oligosaccharides to carboxypeptidase Y and proteinase A in vivo. In addition, in vitro N‐glycosylation of the acceptor peptide Tyr‐Asn‐Leu‐Thr‐Ser‐Val using microsomal membranes from WBP1 depleted cells is reduced as compared with membranes from wild‐type cells. We propose that WBP1 is an essential component of the oligosaccharyl transferase in yeast.

The Oligosaccharyltransferase Complex from Saccharomyces cerevisiae ISOLATION OF THE OST6 GENE, ITS SYNTHETIC INTERACTION WITH OST3, AND ANALYSIS OF THE NATIVE COMPLEX

The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 orOST3 causes only a minor defect inN-glycosylation, but an Δost3Δost6double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteinsin vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a ∼240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation.

Carbohydrate deficient glycoprotein syndrome type IV: deficiency of dolichyl‐P‐Man:Man 5 GlcNAc 2 ‐PP‐dolichyl mannosyltransferase

Type IV of the carbohydrate deficient glycoprotein syndromes (CDGS) is characterized by microcephaly, severe epilepsy, minimal psychomotor development and partial deficiency of sialic acids in serum glycoproteins. Here we show that the molecular defect in the index patient is a missense mutation in the gene encoding the mannosyltransferase that transfers mannose from dolichyl‐phosphate mannose on to the lipid‐linked oligosaccharide (LLO) intermediate Man5GlcNAc2‐PP‐dolichol. The defect results in the accumulation of the LLO intermediate and, due to its leaky nature, a residual formation of full‐length LLOs. N‐glycosylation is abnormal because of the transfer of truncated oligosaccharides in addition to that of full‐length oligosaccharides and because of the incomplete utilization of N‐glycosylation sites. The mannosyltransferase is the structural and functional orthologue of the Saccharomyces cerevisiae ALG3 gene.

Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity.

Asparagine‐linked N‐glycosylation is an essential protein modification occurring in all eukaryotic cells. The central step is the co‐translational transfer of the core oligosaccharide assembled on the lipid carrier dolichol phosphate to selected Asn‐X‐Ser/Thr residues of nascent polypeptide chains in the endoplasmic reticulum. This reaction is catalyzed by the enzyme N‐oligosaccharyl transferase. In yeast, Wbp1p is an essential component of this enzyme. Using a high copy number suppression approach, the SWP1 gene was isolated as an allele specific suppressor of a wbp1 mutation. Swp1p is a 30 kDa type I transmembrane protein and essential for cell viability. Similar to Wbp1p, depletion of Swp1p results in reduced N‐oligosaccharyl transferase activity in vivo and in vitro. Wbp1p and Swp1p can be chemically cross‐linked, suggesting that both proteins are essential constituents of the N‐oligosaccharyl transferase complex.