C. elegans DPY-19 is a C-mannosyltransferase glycosylating thrombospondin repeats.

Among the different types of protein glycosylation, C-mannosylation of tryptophan residues stands out because of the unique linkage formed between sugar and protein. Instead of the typical O- or N-glycosidic linkage, a C-C bond is used for attachment of a single mannose. C-mannose is characteristically found in thrombospondin type 1 repeats and in the WSXWS motif of type I cytokine receptors. The genetic base of the enzymatic activity catalyzing C-mannosylation was not known. Here we demonstrate that Caenorhabditis elegans DPY-19 is a C-mannosyltransferase. DPY-19 exhibits topological and sequential homology to the N-glycan oligosaccharyltransferase, highlighting an evolutionary link between N- and C-glycosylation. We show that the C. elegans surface receptors MIG-21 and UNC-5 are acceptor substrates of DPY-19 and that C-mannosylation is essential for the secretion of soluble MIG-21. Thereby, our data provide an explanation for the previously described identical Q neuroblast migration phenotypes of dpy-19 and mig-21 mutants.

Identification of alg3 in the mushroom-forming fungus Schizophyllum commune and analysis of the Δalg3 knockout mutant.

Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man(5)GlcNAc(2)-PP-Dol resulting in the formation of Man(6)GlcNAc(2)-PP-Dol, which is then further processed to the final precursor oligosaccharide Glc(3)Man(9)GlcNAc(2) for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the Δalg3 strain of S. cerevisiae. Inactivation of alg3 in S. commune resulted in the production of predominantly Man(3)GlcNAc(2) protein-linked N-glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms.

Defining the phenotype in congenital disorder of glycosylation due to ALG1 mutations.

Deficiency of ß-1,4 mannosyltransferase (MT-1) congenital disorder of glycosylation (CDG), due to ALG1 gene mutations. Features in 9 patients reported previously consisted of prenatal growth retardation, pregnancy-induced maternal hypertension and fetal hydrops. Four patients died before 5 years of age, and survivors showed a severe psychomotor retardation. We report on 7 patients with psychomotor delay, microcephaly, strabismus and coagulation abnormalities, seizures and abnormal fat distribution. Four children had a stable clinical course, two had visual impairment, and 1 had hearing loss. Thrombotic and vascular events led to deterioration of the clinical outcome in 2 patients. Four novel ALG1 mutations were identified. Pathogenicity was determined in alg1 yeast mutants transformed with hALG1. Functional analyses showed all novel mutations representing hypomorphs associated with residual enzyme activity. We extend the phenotypic spectrum including the first description of deafness in MT1 deficiency, and report on mildly affected patients, surviving to adulthood. The dysmorphic features, including abnormal fat distribution and strabismus highly resemble CDG due to phosphomannomutase-2 deficiency (PMM2-CDG), the most common type of CDG. We suggest testing for ALG1 mutations in unsolved CDG patients with a type 1 transferrin isoelectric focusing pattern, especially with epilepsy, severe visual loss and hemorrhagic/thrombotic events.

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast.

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as ß-1,6-glucan synthesis, actin cytoskeleton dynamics or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here, we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase (OST) complex, the key enzyme of N-glycosylation. The deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity toward compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas the deletion of OST6, a functional homolog of OST3, has no effect. OST activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared with wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase Y, Gas1 or dipeptidyl aminopeptidase B, displayed an underglycosylation pattern. By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the O-mannosylation process, as the glycosylation of distinct glycoproteins of this type were affected as well. Altogether, the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing.

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included the sequential application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homozygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene identification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous families. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleotide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in the CDG-I in newly diagnosed singleton families.

Autosomal recessive dilated cardiomyopathy due to DOLK mutations results from abnormal dystroglycan O-mannosylation.

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5-13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.

A novel cerebello-ocular syndrome with abnormal glycosylation due to abnormalities in dolichol metabolism.

Cerebellar hypoplasia and slowly progressive ophthalmological symptoms are common features in patients with congenital disorders of glycosylation type I. In a group of patients with congenital disorders of glycosylation type I with unknown aetiology, we have previously described a distinct phenotype with severe, early visual impairment and variable eye malformations, including optic nerve hypoplasia, retinal coloboma, congenital cataract and glaucoma. Some of the symptoms overlapped with the phenotype in other congenital disorders of glycosylation type I subtypes, such as vermis hypoplasia, anaemia, ichtyosiform dermatitis, liver dysfunction and coagulation abnormalities. We recently identified pathogenic mutations in the SRD5A3 gene, encoding steroid 5α-reductase type 3, in a group of patients who presented with this particular phenotype and a common metabolic pattern. Here, we report on the clinical, genetic and metabolic features of 12 patients from nine families with cerebellar ataxia and congenital eye malformations diagnosed with SRD5A3-congenital disorders of glycosylation due to steroid 5α-reductase type 3 defect. This enzyme is necessary for the reduction of polyprenol to dolichol, the lipid anchor for N-glycosylation in the endoplasmic reticulum. Dolichol synthesis is an essential metabolic step in protein glycosylation. The current defect leads to a severely abnormal glycosylation state already in the early phase of the N-glycan biosynthesis pathway in the endoplasmic reticulum. We detected high expression of SRD5A3 in foetal brain tissue, especially in the cerebellum, consistent with the finding of the congenital cerebellar malformations. Based on the overlapping clinical, biochemical and genetic data in this large group of patients with congenital disorders of glycosylation, we define a novel syndrome of cerebellar ataxia associated with congenital eye malformations due to a defect in dolichol metabolism.

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.

A severe human metabolic disease caused by deficiency of the endoplasmatic mannosyltransferase hALG11 leads to congenital disorder of glycosylation-Ip.

A new type of congenital disorders of glycosylation, designated CDG-Ip, is caused by the deficiency of GDP-Man:Man3GlcNAc2-PP-dolichol-alpha1,2-mannosyltransferase, encoded by the human ortholog of ALG11 from yeast. The patient presented with a multisystemic disorder characterized by muscular hypotonia, seizures, developmental retardation and death at the age of 2 years. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains, which is a characteristic sign for CDG-I. Analysis of dolichol-linked oligosaccharides in patient-derived fibroblasts revealed an accumulation of Man3GlcNAc2-PP-dolichol and Man4GlcNAc2-PP-dolichol. Determination of mannosyltransferase activities of early steps of lipid-linked oligosaccharide biosynthesis in fibroblasts indicated that the patient was deficient in elongating Man3GlcNAc2-PP-dolichol. These findings gave rise to genetic analysis of the hALG11 cDNA, in which homozygosity for mutation c.T257C (p.L86S) was identified. Verification of the mutation as a primary cause for the genetic defect was proved by retroviral expression of human wild-type and mutated ALG11 cDNA in patient-derived fibroblasts as well as using a yeast alg11 deletion strain as a heterologous expression system for hALG11 variants. Immunofluorescence examinations combined with western blotting showed no differences of intracellular localization or expression of ALG11 between control and patient fibroblasts, respectively, indicating no mislocalization or degradation of the mutated transferase.

Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an alpha1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide.

The biosynthesis of asparagine-linked glycans occurs in an evolutionarily conserved manner with the assembly of the unique lipid-linked oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol at the ER (endoplasmic reticulum). In the present study we characterize Alg11 from yeast as a mannosyltransferase catalysing the sequential transfer of two alpha1,2-linked mannose residues from GDP-mannose to Man3GlcNAc2-PP-Dol and subsequently to Man4GlcNAc2-PP-Dol forming the Man5GlcNAc2-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side. Alg11 is predicted to contain three hydrophobic transmembrane-spanning helices. Using Alg11 topology reporter fusion constructs, we show that only the N-terminal domain fulfils this criterion. Surprisingly, this domain can be deleted without disturbing glycosyltransferase function and membrane association, indicating also that the other two hydrophobic domains contribute to ER localization, but in a non-transmembrane manner. By site-directed mutagenesis we investigated amino acids important for transferase activity. We demonstrate that the first glutamate residue in the EX7E motif, conserved in a variety of glycosyltransferases, is more critical than the second, and loss of Alg11 function occurs only when both glutamate residues are exchanged, or when the mutation of the first glutamate residue is combined with replacement of another amino acid in the motif. This indicates that perturbations in EX7E are not restricted to the second glutamate residue. Moreover, Gly85 and Gly87, within a glycine-rich domain as part of a potential flexible loop, were found to be required for Alg11 function. Similarly, a conserved lysine residue, Lys319, was identified as being important for activity, which could be involved in the binding of the phosphate of the glycosyl donor.

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.

Biochemical characterization and membrane topology of Alg2 from Saccharomyces cerevisiae as a bifunctional alpha1,3- and 1,6-mannosyltransferase involved in lipid-linked oligosaccharide biosynthesis.

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc(3)Man(9) GlcNAc(2)-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the alpha1,3- and the alpha1,6-mannose-linked residue from GDP-mannose to Man(1)GlcNAc(2)-PP-Dol forming the Man(3)GlcNAc(2)-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX(7)E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys(230), as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.

The yeast oligosaccharyltransferase complex can be replaced by STT3 from Leishmania major.

The key step of protein N-glycosylation is catalyzed by the multimeric oligosaccharyltransferase complex (OST). Biochemical and genetic studies have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1, Swp1, Stt3, Ost1, Ost2, Ost3, Ost4, Ost5, and Ost6. With the exception of Stt3, assumed to contain the catalytic site, little is known about the function of other OST subunits. The existence of the OST complex is suggested to allow substrate specificity and efficient transfer, a close interaction with the translocon and the prevention of protein folding to ensure the efficient co-translational modification of proteins. However, in the recently completed genome of the trypanosomatid parasite Leishmania major STT3 (of which four paralogs exist, STT3-1, STT3-2, STT3-3, and STT3-4) is the only OST subunit that can be identified. Here we report that L.m.STT3 proteins, except STT3-3, are able to complement stt3 deficiency in yeast during vegetative growth, but only poorly during sporulation. By blue native electrophoresis we demonstrate that the L.mSTT3 is active mainly as a free, monomeric enzyme. In cell-free assays and also in vivo we find that L.mSTT3, expressed in yeast, has a broad specificity for nonglucosylated lipid-linked mannose-oligosaccharides, typical for several protists. But when incorporated into the OST complex, L.mSTT3 transfers also the common eukaryotic Glc(3)Man(9)GlcNAc(2)-PP-Dol donor. Finally, three L.m.STT3 paralogs were shown to complement not only stt3 but also ost1, ost2, wbp1, or swp1 mutants. Thus, STT3 from Leishmania can substitute for the whole OST complex.

Identification of the gene encoding the alpha1,3-mannosyltransferase (ALG3) in Arabidopsis and characterization of downstream n-glycan processing.

Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a mannosyltransferase gene from Arabidopsis thaliana, which is the functional homolog of the ALG3 (Dol-P-Man:Man5GlcNAc2-PP-Dol alpha1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a Deltaalg3 yeast mutant and is localized to the endoplasmic reticulum in yeast and in plants. A homozygous T-DNA insertion mutant, alg3-2, was identified in Arabidopsis with residual levels of wild-type ALG3, derived from incidental splicing of the 11th intron carrying the T-DNAs. N-glycan analysis of alg3-2 and alg3-2 in the complex-glycan-less mutant background, which lacks N-acetylglucosaminyl-transferase I activity, reveals that when ALG3 activity is strongly reduced, almost all N-glycans transferred to proteins are aberrant, indicating that the Arabidopsis oligosaccharide transferase complex is remarkably substrate tolerant. In alg3-2 plants, the aberrant glycans on glycoproteins are recognized by endogenous mannosidase I and N-acetylglucosaminyltransferase I and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3-2 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response.

Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases.

Proteins can be modified by a large variety of covalently linked saccharides. The present review concentrates on two types, protein N-glycosylation and protein O-mannosylation, which, with only a few exceptions, are evolutionary conserved from yeast to man. They are also distinguished by some special features: The corresponding glycosylation processes start in the endoplasmatic reticulum, are continued in the Golgi apparatus, and require dolichol-activated precursors for the initial biosynthetic steps. With respect to the molecular biology of both types of protein glycosylation, the pathways and the genetic background of the reactions have most successfully been studied with the genetically easy-to-handle baker's yeast, Saccharomyces cerevisae. Many of the severe developmental disturbances in children are related to protein glycosylation, for example, the CDG syndrome (congenital disorders of glycosylation) as well as congenital muscular dystrophies with neuronal-cell-migration defects have been elucidated with the help of yeast.

Pir proteins of Saccharomyces cerevisiae are attached to beta-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 gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

Defects in N-glycosylation induce apoptosis in yeast.

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Defects of N-glycosylation in humans lead to congenital disorders. The pivotal step of this pathway is the transfer of the evolutionarily conserved lipid-linked core-oligosaccharide to the nascent polypeptide chain, catalysed by the oligosaccharyltransferase. One of its nine subunits, Ost2, has homology to DAD1, originally characterized in hamster cells as a defender against apoptotic death. Here we show that ost mutants, such as ost2 and wbp1-1, display morphological and biochemical features of apoptosis upon induction of the glycosylation defect. We observe nuclear condensation, DNA fragmentation as well as externalization of phosphatidylserine. We also demonstrate induction of caspase-like activity, both determined by flow cytometric analysis and in cell-free extracts. Similarly, the N-glycosylation inhibitor tunicamycin in combination with elevated temperature is able to challenge the apoptotic cascade. Heterologous expression of anti-apoptotic human Bcl-2 diminishes caspase activation, improves survival of cells and suppresses the temperature-sensitive growth defect of wbp1-1. Furthermore, accumulation of reactive oxygen species occurs in response to defective glycosylation. As deletion of the metacaspase YCA1 does not seem to abrogate glycosylation-induced apoptosis, we postulate a different proteolytic process to be involved in this death pathway.

Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit.

The key step of N-glycosylation of proteins in the endoplasmic reticulum is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). It transfers the lipid-linked core-oligosaccharide to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains. Biochemical and genetic approaches have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1p, Swp1p, Stt3p, Ost1p, Ost2p, Ost4p, Ost5p, Ostp3 and Ost6p. By blue native polyacrylamide electrophoresis we show that yeast OST consists of two isoforms with distinct functions differing only in the presence of the two related Ost3 and Ost6p proteins. The OST6-complex was found to be important for cell wall integrity and temperature stress. Ost3p and Ost6p are not essential for OST activity, and can in part displace each other in the complex when overexpressed, suggesting a dynamic regulation of the complex formation.