Golgi Mannosyltransferases Research Articles

The role of manganese in morphogenesis and pathogenesis of the opportunistic fungal pathogen Candida albicans.

Metals such as Fe, Cu, Zn, and Mn are essential trace nutrients for all kingdoms of life, including microbial pathogens and their hosts. During infection, the mammalian host attempts to starve invading microbes of these micronutrients through responses collectively known as nutritional immunity. Nutritional immunity for Zn, Fe and Cu has been well documented for fungal infections; however Mn handling at the host-fungal pathogen interface remains largely unexplored. This work establishes the foundation of fungal resistance against Mn associated nutritional immunity through the characterization of NRAMP divalent metal transporters in the opportunistic fungal pathogen, Candida albicans. Here, we identify C. albicans Smf12 and Smf13 as two NRAMP transporters required for cellular Mn accumulation. Single or combined smf12Δ/Δ and smf13Δ/Δ mutations result in a 10-80 fold reduction in cellular Mn with an additive effect of double mutations and no losses in cellular Cu, Fe or Zn. As a result of low cellular Mn, the mutants exhibit impaired activity of mitochondrial Mn-superoxide dismutase 2 (Sod2) and cytosolic Mn-Sod3 but no defects in cytosolic Cu/Zn-Sod1 activity. Mn is also required for activity of Golgi mannosyltransferases, and smf12Δ/Δ and smf13Δ/Δ mutants show a dramatic loss in cell surface phosphomannan and in glycosylation of proteins, including an intracellular acid phosphatase and a cell wall Cu-only Sod5 that is key for oxidative stress resistance. Importantly, smf12Δ/Δ and smf13Δ/Δ mutants are defective in formation of hyphal filaments, a deficiency rescuable by supplemental Mn. In a disseminated mouse model for candidiasis where kidney is the primary target tissue, we find a marked loss in total kidney Mn during fungal invasion, implying host restriction of Mn. In this model, smf12Δ/Δ and smf13Δ/Δ C. albicans mutants displayed a significant loss in virulence. These studies establish a role for Mn in Candida pathogenesis.

The Mnn10/Anp1-dependent N-linked outer chain glycan is dispensable for Candida albicans cell wall integrity.

Candida albicans cell wall glycoproteins, and in particular their mannose-rich glycans, are important for maintaining cellular integrity as well as host recognition, adhesion, and immunomodulation. The asparagine (N)-linked mannose outer chain of these glycoproteins is produced by Golgi mannosyltransferases (MTases). The outer chain is composed of a linear backbone of ∼50 α1,6-linked mannoses, which acts as a scaffold for addition of ∼150 or more mannoses in other linkages. Here, we describe the characterization of C. albicans OCH1, MNN9, VAN1, ANP1, MNN10, and MNN11, which encode the conserved Golgi MTases that sequentially catalyze the α1,6 mannose outer chain backbone. Candida albicans och1Δ/Δ, mnn9Δ/Δ, and van1Δ/Δ mutants block the earliest steps of backbone synthesis and like their Saccharomyces cerevisiae counterparts, have severe cell wall and growth phenotypes. Unexpectedly, and in stark contrast to S. cerevisiae, loss of Anp1, Mnn10, or Mnn11, which together synthesize most of the backbone, have no obvious deleterious phenotypes. These mutants were unaffected in cell morphology, growth, drug sensitivities, hyphal formation, and macrophage recognition. Analyses of secreted glycosylation reporters demonstrated that anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ strains accumulate glycoproteins with severely truncated N-glycan chains. This hypo-mannosylation did not elicit increased chitin deposition in the cell wall, which in other yeast and fungi is a key compensatory response to cell wall integrity breaches. Thus, C. albicans has evolved an alternate mechanism to adapt to cell wall weakness when N-linked mannan levels are reduced.

N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a robust host for heterologous protein expression. The efficient expression of cellulases in S. cerevisiae is important for the consolidated bioprocess that directly converts lignocellulose into valuable products. However, heterologous proteins are often N-hyperglycosylated in S. cerevisiae, which may affect protein activity. In this study, the expression of three heterologous proteins, β-glucosidase, endoglucanase and cellobiohydrolase, was found to be N-hyperglycosylated in S. cerevisiae. To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Och1p, Mnn9p and Mnn1p), respectively. Their extracellular activities improved markedly in the OCH1 and MNN9 deletion strains. Interestingly, truncation of the N-hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield. Further analysis showed OCH1 and MNN9 deletion up-regulated genes in the secretory pathway, such as protein folding and vesicular trafficking, but did not induce the unfolded protein response. The cell wall integrity was also affected by OCH1 and MNN9 deletion, which contributed to the release of secretory protein extracellularly. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion.

Sac1-Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus.

Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot-Marie-Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1-Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

Svp26 Facilitates Endoplasmic Reticulum to Golgi Transport of a Set of Mannosyltransferases in Saccharomyces cerevisiae

Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the Deltasvp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26.

The Yeast LATS/Ndr Kinase Cbk1 Regulates Growth via Golgi-dependent Glycosylation and Secretion

Saccharomyces cerevisiae Cbk1 is a LATS/Ndr protein kinase and a downstream component of the regulation of Ace2 and morphogenesis (RAM) signaling network. Cbk1 and the RAM network are required for cellular morphogenesis, cell separation, and maintenance of cell integrity. Here, we examine the phenotypes of conditional cbk1 mutants to determine the essential function of Cbk1. Cbk1 inhibition severely disrupts growth and protein secretion, and triggers the Swe1-dependent morphogenesis checkpoint. Cbk1 inhibition also delays the polarity establishment of the exocytosis regulators Rab-GTPase Sec4 and its exchange factor Sec2, but it does not interfere with actin polarity establishment. Cbk1 binds to and phosphorylates Sec2, suggesting that it regulates Sec4-dependent exocytosis. Intriguingly, Cbk1 inhibition causes a >30% decrease in post-Golgi vesicle accumulation in late secretion mutants, indicating that Cbk1 also functions upstream of Sec2-Sec4, perhaps at the level of the Golgi. In agreement, conditional cbk1 mutants mislocalize the cis-Golgi mannosyltransferase Och1, are hypersensitive to the aminoglycoside hygromycin B, and exhibit diminished invertase and Sim1 glycosylation. Significantly, the conditional lethality and hygromycin B sensitivity of cbk1 mutants are suppressed by moderate overexpression of several Golgi mannosyltransferases. These data suggest that an important function for Cbk1 and the RAM signaling network is to regulate growth and secretion via Golgi and Sec2/Sec4-dependent processes.

Golgi Localization of Glycosyltransferases Requires a Vps74p Oligomer

The mechanism of glycosyltransferase localization to the Golgi apparatus is a long-standing question in secretory cell biology. All Golgi glycosyltransferases are type II membrane proteins with small cytosolic domains that contribute to Golgi localization. To date, no protein has been identified that recognizes the cytosolic domains of Golgi enzymes and contributes to their localization. Here, we report that yeast Vps74p directly binds to the cytosolic domains of cis and medial Golgi mannosyltransferases and that loss of this interaction correlates with loss of Golgi localization of these enzymes. We have solved the X-ray crystal structure of Vps74p and find that it forms a tetramer, which we also observe in solution. Deletion of a critical structural motif disrupts tetramer formation and results in loss of Vps74p localization and function. Vps74p is highly homologous to the human GMx33 Golgi matrix proteins, suggesting a conserved function for these proteins in the Golgi enzyme localization machinery.

Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

Retrograde Transport of the Mannosyltransferase Och1p to the Early Golgi Requires a Component of the COG Transport Complex

The yeast COG complex has been proposed to function as a vesicle-tethering complex on an early Golgi compartment, but its role is not fully understood. COG complex mutants exhibit a dramatic reduction in Golgi-specific glycosylation and other defects. Here we show that a strain carrying a COG3 temperature-sensitive allele, cog3-202, clearly exhibited the glycosylation defect while exhibiting nearly normal secretion kinetics. Two Golgi mannosyltransferases, Och1p and Mnn1p, were mislocalized in cog3-202 cells. In cog3-202 cells Och1-HA was found in lighter density membranes than in wild type cells. In sed5(ts) and sft1(ts) strains, Och1p rapidly accumulated in vesicle-like structures consistent with the delivery of Och1p back to the cis-Golgi on retrograde vesicles via a Sed5p/Sft1p-containing SNARE complex. In contrast to cog3-202 cells, the membranes in sed5(ts) cells that contained Och1p were denser than in wild type. Together these results indicate that Och1p does not accumulate in retrograde vesicles in the cog3-202 mutant and are consistent with the COG complex playing a role in sorting of Och1p into retrograde vesicles. In wild type cells Och1p has been shown previously to cycle between the cis-Golgi and minimally as far as the late Golgi. We find that Och1p does not cycle via endosomes during its normal itinerary suggesting that Och1p engages in intra-Golgi cycling only. However, Och1p does use a post-Golgi pathway for degradation because a portion of Och1p was degraded in the vacuole. Most surprisingly, Och1p can use either the carboxypeptidase Y or AP-3 pathways to reach the vacuole for degradation.

Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum.

In yeast primary N- and O-glycans are attached to proteins in the endoplasmic reticulum (ER), and they are elongated in the Golgi. Thus, glycan extension by Golgi enzymes has been taken as evidence for arrival of a protein in the Golgi. Two alpha 1,6-mannosyltransferase activity-containing multiprotein complexes have been reported to recycle between the Golgi and the ER, but since resident ER proteins are not Golgi-modified, Golgi enzymes were not thought to function in the ER. Here we show that when protein exit from the ER was blocked in COPII-defective yeast mutants, the N-glycans of vacuolar carboxypeptidase Y and a set of unidentified glycoproteins were decorated with an alpha 1,6-mannose residue, normally added in the Golgi by Och1p. Immunofluorescent staining demonstrated that Och1p accumulated in the ER under these conditions. Concomitantly, primary O-glycans of a secretory protein were extended, apparently by the medial Golgi transferase Mnt1p. Similar O-glycan extension occurred in wild-type cells when an HDEL-tagged protein was allowed to encounter glycosyltransferases in the Golgi during recycling between ER and Golgi. Golgi-specific glycosylation in the ER was reduced when Golgi-to-ER traffic was blocked, confirming that glycan extension in the ER was mainly due to recycling, rather than newly synthesized transferases.

Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum.

Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have alpha-1,6-mannosyltransferase activity in vitro. In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells. This behavior is consistent with an active Golgi to ER recycling process. To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay. Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.

Proteins in the early Golgi compartment of Saccharomyces cerevisiae immunoisolated by Sed5p

The yeast tSNARE Sed5p is considered to mainly reside in the early Golgi compartment at the steady state of its intracellular cycling. To better understand this compartment, we immunoisolated a membrane subfraction having Sed5p on the surface (the Sed5 vesicles). Immunoblot studies showed that considerable portions (20–30%) of the Golgi mannosyltransferases (Mnt1p, Van1p, and Mnn9p) were simultaneously recovered while the late Golgi (Kex2p) or endoplasmic reticulum (Sec71p) proteins were almost excluded. The N-terminal sequences of the polypeptides detectable by Coomassie blue staining indicated that the prominent components of the Sed5 vesicles include Anp1p, Emp24p, Erv25p, Erp1p, Ypt52p, and a putative membrane protein of unknown function (Yml067c).

Activity of the yeast MNN1 alpha-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases.

A wide diversity of biological molecules are modified by the addition of sugar residues, and a large number of glycosyltransferases have been identified that are responsible for these reactions. Despite catalyzing closely related reactions, many of these transferases show little apparent sequence homology. By comparing two apparently unrelated families of yeast Golgi mannosyltransferases, a short motif containing two aspartate residues was observed that was conserved in both groups of proteins. Mutagenesis of one of the members of these families, the alpha-1, 3-mannosyltransferase Mnn1p, showed that altering either of these aspartates eliminates all enzymatic activity. These changes do not appear to affect the overall folding and assembly of Mnn1p. A similar aspartate-containing sequence was found to be conserved in a diverse range of other glycosyltransferase families, much more frequently than would be expected by chance, suggesting that it is a feature of the catalytic site, or an element of a structural fold, shared by many glycosyltransferases.

Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport.

To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Och1p, a cis-Golgi mannosyltransferase. A series of influenza virus hemagglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-length Och1p. Several constructs included a Kex2p cleavage site between the Och1p and invertase moieties to monitor transit to the Kex2p-containing TGN. Cells expressing an Och1p-invertase fusion do not secrete invertase, but those expressing an Och1p-Kex2p site-invertase fusion protein secrete high levels of invertase in a Kex2p-dependent manner. The Och1p-Kex2p site-invertase fusion protein is cleaved with a half-time of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage through the medial- and trans-Golgi, therefore cleavage occurs after ordered anterograde transport through the Golgi to the TGN. Transit to distal compartments is not induced by the invertase moiety, since noninvertase fusion constructs encounter the same glycosyltransferases and Kex2p as well. The Och1p-HA moiety, irrespective of whether it is generated by cleavage of the fusion protein in the TGN or synthesized de novo, is degraded with a half-time of about 60 min. Thus, the half-time of degradation is 12-fold longer than the time required to reach the TGN. At steady state, de novo-synthesized and TGN-generated HA epitope-tagged Och1p reside in a compartment with a buoyant density identical to that of wild-type Och1p and distinct from that of the vacuole or the TGN. Finally, och1 null cells that express an Ochlp fusion construct known to rapidly encounter the TGN glycosylate invertase to the same extent as wild-type cells, indicating that they have phenotypically wild-type Och1p activity. These results lead us to propose a model for Och1p-HA localization that involves movement to distal compartments, at least as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.

Isolation of protein glycosylation mutants in the fission yeast Schizosaccharomyces pombe.

We have isolated mutants in the fission yeast Schizosaccharomyces pombe that are defective in protein glycosylation. A collection of osmotically sensitive mutants was prepared and screened for glycosylation defects using lectin staining as an assay. Mutants singly defective in four glycoprotein synthesis genes (gps1-4) were isolated, all of which bind less galactose-specific lectin. Acid phosphatase and other glycoproteins from the gps mutants have increased electrophoretic mobility, suggesting that these mutants make glycans of reduced size. N-linked glycan analysis revealed that terminal oligosaccharide modification is defective in the gps1 and gps2 mutants. Both mutants synthesize the Man9GlcNAc2 core glycan but have reduced amounts of larger structures. Modified core glycans from gps1 cells have normal amounts of galactose (Gal) residues, but reduced amounts of Man, consistent with a defect in a Golgi mannosyltransferase in this mutant. In contrast, N-linked oligosaccharides from gps2 mutants have much less Gal than wild type, because of reduced levels of the Gal donor, UDP-Gal. This reduction is caused by decreased activity of UDP-glucose 4-epimerase, which synthesizes UDP-Gal. Neither the gps1 or gps2 mutations are lethal, although the cells grow at reduced rates. These findings suggest that S. pombe cells can survive with incompletely glycosylated cell wall glycoproteins. In particular, these results suggest that Gal, which comprises approximately 30% by weight of cell wall glycoprotein glycans, is not crucial for cell growth or survival.

Mannosyltransferase activities in membranes from various yeast strains.

In the yeast Golgi compartments, at least five, and potentially several additional mannosyltransferases are involved in elongating to 'mannan' the core Man8GlcNAc2 oligosaccharide trimmed from Glc3Man9GlcNAc2 in the endoplasmic reticulum. Structural studies on oligosaccharides from alg3 mutant yeast, which lack the four upper arm mannoses donated by Man-P-Dol (where Dol is dolichol), verified that the new alpha 1,6-branch in endo H-resistant mannan in this strain is efficiently initiated in vivo on the alpha 1,3-linked core residue of the lipid-oligosaccharide form of Man5GlcNAc2 (Verostek et al., J. Biol. Chem., 266, 5547-5551, 1991). This Man5GlcNAcGlcNAc[3H]ol isomer (where GlcNAc[3H]ol is N-acetylglucosamin [1-3H] itol) was found to be an excellent acceptor for a number of GDP-Man-dependent Golgi mannosyltransferases in detergent-solubilized yeast membrane preparations: an alpha 1,3-mannosyltransferase (Mnn1p), an alpha 1,6-mannosyltransferase (Och1p) and two alpha 1,2-mannosyltransferases (Mnt1p/Kre2p,?) whose products were readily identified by 1H NMR spectroscopy. The Man6GlcNAcGlcNAc[3H]ol isomers formed were easily defined by alpha 1,2-mannosidase sensitivity and either Bio-Gel P-4 gel filtration or AX-5 high-performance liquid chromatography. In general, mannosyltransferases present in detergent-solubilized microsomes from most yeast strains mimicked the array of sugar linkages observed on their respective glycoproteins. However, in the case of the Saccharomyces pmr1 mutant, an alpha 1,3-mannosyltransferase was active in microsomal extracts, but the alpha 1,3-Man epitope could not be identified on Western blots of cellular glycoproteins using sugar linkage-specific antibodies or lectins. The in vitro transferase assay is simple, rapid and accurate, and in the case of pmr1 suggests that in vivo either invertase is misrouted during secretion or the alpha 1,3-mannosyltransferase is mistargeted after its synthesis in this mutant.

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.

Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an α-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.