Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of alpha-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides.

The genome of Saccharomyces cerevisiae contains five genes that encode type II transmembrane proteins with significant amino acid similarity to the alpha-1,3-mannosyltransferase Mnn1p. The roles of the three genes most closely related to MNN1 were examined in mutants carrying single and multiple combinations of the disrupted genes. Paper chromatographic analysis of [2-3H]mannose-labeled O-linked oligosaccharides released by beta-elimination showed that the MNT2 (YGL257c) and MNT3 (YIL014w) genes in combination with MNN1 have overlapping roles in the addition of the fourth and fifth alpha-1,3-linked mannose residues to form Man4 and Man5 oligosaccharides whereas MNT4 (YNR059w) does not appear to be required for O-glycan synthesis.

The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae.

Glycosylation constitutes one of the most important of all the post-translational modifications and may have numerous effects on the function, structure, physical properties and targeting of particular proteins. Eukaryotic glycan structures are progressively elaborated in the secretory pathway. Following the addition of a core N-linked carbohydrate in the endoplasmic reticulum, glycoproteins move to the Golgi complex where the elongation of O-linked sugar chains and processing of complex N-linked oligosaccharide structures take place. In order to better define how such post-translational modifications occur, we have been studying the yeast KTR and MNN1 mannosyltransferase gene families. The KTR family contains nine members: KRE2, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6 and KTR7. The MNN1 family contains six members: MNN1, TTP1, YGL257c, YNR059w, YIL014w and YJL86w. In this review, we address protein structure, sequence similarities and enzymatic activity in the context of each gene family. In addition, a description of the known function of many family members in O- and N-linked glycosylation is included. Finally, the genetic interactions and functional redundancies within a gene family are also discussed.

The Candida albicans KRE9 gene is required for cell wall beta-1, 6-glucan synthesis and is essential for growth on glucose.

We have isolated CaKRE9, a gene from Candida albicans, that is a functional homologue of the Saccharomyces cerevisiae KRE9 gene involved in beta-1,6-glucan synthesis. Disruption of the CaKRE9 gene in C. albicans shows that CaKre9p is required for the synthesis or assembly of this fungal polymer. Homozygous null disruptants of CaKRE9 grow poorly on galactose and fail to form hyphae in serum, and, in growth medium containing glucose, the gene is essential. Thus, the CaKRE9 gene product is a potentially useful candidate as a target for fungal-specific drugs.

Involvement of protein N-glycosyl chain glucosylation and processing in the biosynthesis of cell wall beta-1,6-glucan of Saccharomyces cerevisiae.

beta-1,6-Glucan plays a key structural role in the yeast cell wall. Of the genes involved in its biosynthesis, the activity of Cwh41p is known, i.e., the glucosidase I enzyme of protein N-chain glucose processing. We therefore examined the effects of N-chain glucosylation and processing mutants on beta-1,6-glucan biosynthesis and show that incomplete N-chain glucose processing results in a loss of beta-1,6-glucan, demonstrating a relationship between N-chain glucosylation/processing and beta-1,6-glucan biosynthesis. To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, we investigated the Saccharomyces cerevisiae KRE5 and CNE1 genes, which encode homologs of the "quality control" components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively. We show that the essential activity of Kre5p is separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase. We also observe a approximately 30% decrease in beta-1,6-glucan upon disruption of the CNE1 gene, a phenotype that is additive with other beta-1,6-glucan synthetic mutants. Analysis of the cell wall anchorage of the mannoprotein alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification.

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains.

We have determined a role for Ktr1p and Ktr3p as mannosyltransferases in the synthesis of the carbohydrate chains attached to Saccharomyces cerevisiae O- and N-modified proteins. KTR1 and KTR3 encode related proteins that are highly similar to the Kre2p/Mnt1p Golgi alpha1,2-mannosyltransferase (Lussier, M., Camirand, A., Sdicu, A.-M., and Bussey, H. (1993) Yeast 9, 1057-1063; Mallet, L., Bussereau, F., and Jacquet, M. (1994) Yeast 10, 819-831). Examination of the electrophoretic mobility of a specifically O-linked protein from mutants and an analysis of their total O-linked mannosyl chains demonstrates that Ktr1p, Ktr3p, and Kre2p/Mnt1p have overlapping roles and collectively add most of the second and the third alpha1,2-linked mannose residues on O-linked oligosaccharides. Determination of the mobility of the specifically N-linked glycoprotein invertase in different null strains indicates that Ktr1p, Ktr3p, and Kre2p are also jointly involved in N-linked glycosylation, possibly in establishing some of the outer chain alpha1,2-linkages.

Completion of the Saccharomyces cerevisiae genome sequence allows identification of KTR5, KTR6 and KTR7 and definition of the nine-membered KRE2/MNT1 mannosyltransferase gene family in this organism.

The KRE2/MNT1 mannosyltransferase gene family of Saccharomyces cerevisiae currently consists of the KRE2, YUR1, KTR1, KTR2, KTR3 and KTR4 genes. All six encode putative type II membrane proteins with a short cytoplasmic N-terminus, a membrane-spanning region and a highly conserved catalytic lumenal domain. Here we report the identification of the three remaining members of this family in the yeast genome. KTR5 corresponds to an open reading frame (ORF) of the left arm of chromosome XIV, and KTR6 and KTR7 to ORFs on the left arms of chromosomes XVI and IX respectively. The KTR5, KTR6 and KTR7 gene products are highly similar to the Kre2p/Mnt1p family members. Initial functional characterization revealed that some mutant yeast strains containing null copies of these genes displayed cell wall phenotypes. None was K1 killer toxin resistant but ktr6 and ktr7 null mutants were found to be hypersensitive and resistant, respectively, to the drug Calcofluor White.

Ktr1p is an alpha-1,2-mannosyltransferase of Saccharomyces cerevisiae. Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris.

The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the alpha 1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied. In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to those of Kre2p/Mnt1p. Recombinant soluble forms of Kre2p/Mnt1p and Ktr1p lacking their N-terminal regions were expressed as secreted proteins from the methylotrophic yeast Pichia pastoris. After induction with methanol, the medium contained approx, 40 and 400 mg/l of soluble recombinant Kre2p/Mnt1p and Ktr1p respectively. Both recombinant proteins were shown to exhibit alpha 1,2-mannosyltransferase activity. The enzymes have an absolute requirement for Mn2+ and a similar K(m) for mannose (280-350 mM), methyl-alpha-mannoside (60-90 mM) and GDP-mannose (50-90 microM), but the Vmax was approx. 10 times higher for Kre2p/Mnt1p than for Ktr1p. The enzymes have similar substrate specificities and utilize mannose, methyl-alpha-mannoside, alpha-1,2-mannobiose and methyl-alpha-1,2-mannobiose, as well as Man15-30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates. The enzymes do not utilize alpha-1,6-mannobiose, alpha-1,6-mannotriose, alpha-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9-10GlcNAc. These results indicate that Kre2p/ Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.

Functional characterization of the YUR1, KTR1, and KTR2 genes as members of the yeast KRE2/MNT1 mannosyltransferase gene family.

Eukaryotic glycan structures are progressively elaborated in the secretory pathway. Following the addition of a core N-linked carbohydrate in the endoplasmic reticulum, glycoproteins move to the Golgi complex where the elongation of O-linked sugar chains and processing of complex N-linked oligosaccharide structures take place. In order to better define how such post-translational modifications occur, we have been studying a yeast gene family in which at least one member, KRE2/MNT1, is involved in protein glycosylation. The family currently contains five other members: YUR1, KTR1, KTR2, KTR3 and KTR4 (Mallet, L., Bussereau, F., and Jacquet, M. (1994) Yeast 10, 819-831). All encode putative type II membrane proteins with a short cytoplasmic N terminus, a membrane-spanning region, and a highly conserved catalytic lumenal domain. Kre2p/Mnt1p is a alpha 1,2-mannosyltransferase involved in O- and N-linked glycosylation (Häusler, A., Ballou, L., Ballou, C.E., and Robbins, P.W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6846-6850); however, the role of the other proteins has not yet been established. We have carried out a functional analysis of Ktr1p, Ktr2p, and Yur1p. By in vitro assays, Ktr1p, Ktr2p, and Yur1p have been shown to be mannosyltransferase but, in vivo, do not appear to be involved in O-glycosylation. Examination of the electrophoretic mobility of the N-linked modified protein invertase in null mutant strains indicates that Ktr1p, Ktr2p, and Yur1p are involved in N-linked glycosylation, possibly as redundant enzymes. As found with Kre2p (Hill, K., Boone, C., Goebl, M., Puccia, R., Sdicu, A.-M., and Bussey, H. (1992) Genetics 130, 273-283), Ktr1p, Ktr2p, and Yur1p also seem to be implicated in the glycosylation of cell wall mannoproteins, since yeast cells containing different gene disruptions become K1 killer toxin-resistant. Immunofluorescence microscopy reveals that like Kre2p; Ktr1p, Ktr2p and Yur1p are localized in the Golgi complex.

Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.

Protein O-glycosylation in yeast. The PMT2 gene specifies a second protein O-mannosyltransferase that functions in addition to the PMT1-encoded activity.

The PMT2 gene from Saccharomyces cerevisiae was identified as FUN25, a transcribed open reading frame on the left arm of chromosome I (Ouellette, B. F. F., Clark, M. W. C., Keng, T., Storms, R. G., Zhong, W., Zeng, B., Fortin, N., Delaney, S., Barton, A., Kaback, D.B., and Bussey, H. (1993) Genome 36, 32-42). The product encoded by the PMT2 gene shows significant similarity with the dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase, Pmt1p (EC 2.4.1.109), which is required for initiating the assembly of O-linked oligosaccharides in S. cerevisiae (Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8164-8168). The PMT2 gene encodes a new protein O-D-mannosyltransferase. Yeast cells carrying a PMT2 disruption show a diminished in vitro and in vivo O-mannosylation activity and resemble mutants with a nonfunctional PMT1 gene. Strains bearing a pmt1 pmt2 double disruption show a severe growth defect but retain residual O-mannosylation activity indicating the presence of at least one more protein-O-mannosyltransferase.

KTR2: a new member of the KRE2 mannosyltransferase gene family.

The KTR2 gene from Saccharomyces cerevisiae was identified by polymerase chain reaction amplification of genomic DNA using primers derived from regions of high homology between the products of three yeast genes, KRE2, YUR1 and KTR1. The product encoded by the KTR2 gene is a predicted type II membrane protein of 425 amino acid residues with a short cytoplasmic N-terminus, a membrane-spanning region and a large lumenal domain containing residues with a short cytoplasmic N-terminus, a membrane-spanning region and a large lumenal domain containing four potential N-glycosylation sites. Ktr2p has 58% identity with Yur1p, 39% with Ktr1p and 34% with Kre2p. One member of this gene family, KRE2 (also known as MNT1; Häusler and Robbins, 1992), encodes an alpha-1,2 mannosyltransferase which adds the third mannose onto O-linked glycoprotein side-chains (Häusler et al., 1992). In contrast to KRE2 null mutants, which produce shortened (two-mannose) chains, mutants harboring a KTR2 gene disruption synthesize O-linked chains with the wild-type patterns of five mannose residues. A null mutation in KTR2 leads to partial resistance to killer toxin and hints that KTR2, which encodes a putative mannosyltransferase, is involved in extracellular matrix assembly.

Yeast KRE2 defines a new gene family encoding probable secretory proteins, and is required for the correct N-glycosylation of proteins.

We have cloned, sequenced and disrupted the KRE2 gene of Saccharomyces cerevisiae, identified by killer-resistant mutants with a defective cell wall receptor for the toxin. The KRE2 gene is close to PHO8 on chromosome 4, and encodes a predicted 49-kD protein, Kre2p, that probably enters the secretory pathway. Haploid cells carrying a disruption of the KRE2 locus grow more slowly than wild-type cells at 30 degrees, and fail to grow at 37 degrees. At 30 degrees, kre2 mutants showed altered N-linked glycosylation of proteins, as the average size of N-linked outer chains was reduced. We identified two other genes, YUR1 on chromosome 10, and KTR1 on chromosome 15, whose predicted products share 36% identity with Kre2p over more than 300 amino acid residues. Yur1p has an N-terminal signal sequence like Kre2p, while Ktr1p has a predicted topology consistent with a type 2 membrane protein. In all cases the conserved regions of these proteins appear to be on the lumenal side of secretory compartments, suggesting related function. KRE2, KTR1 and YUR1 define a new yeast gene family.

Mutual antagonism among killer yeasts: competition between K1 and K2 killers and a novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae.

Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stable in vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 degrees C. At pH 4.0, closer to the in vitro pH optimum of the K2 killer toxin, the K1 strain again predominated at 18 degrees C, but at 30 degrees C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1-K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1-K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.