The Ktr1p, Ktr3p, and Kre2p/Mnt1p Mannosyltransferases Participate in the Elaboration of Yeast O- andN-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- andN-modified proteins. KTR1 and KTR3encode related proteins that are highly similar to the Kre2p/Mnt1p Golgi α1,2-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 α1,2-linked mannose residues onO-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 α1,2-linkages.

Nascent proteins synthesized by membrane-bound ribosomes are translocated across the ER 1 membrane and acquire carbohydrate chains on specific serine, threonine, and asparagine residues. These glycoproteins then proceed through the Golgi complex where their oligosaccharides are further modified. The O-modified proteins of Saccharomyces cerevisiae possess a linear carbohydrate chain of up to 5 mannose residues (1,2). O-Glycosylation is initiated in the ER with dolichyl phosphate-D-mannose as the immediate sugar donor for the mannosyl residue transferred to the hydroxy-amino acids serine and threonine (2,3). GDP-Man is utilized as the sugar donor in the subsequent elongation of O-linked carbohydrate chains resulting in a linear glycan in which the second and third mannoses possess ␣1,2-linkages, whereas the terminal fourth and fifth mannosyl residues have ␣1,3-linkages (1,2).
Yeast N-linked modified proteins can acquire two different types of glycan chains after the addition of a Man 8 GlcNAc 2 core in the endoplasmic reticulum, a process common to the major-ity of eukaryotes (1,2). This primary oligosaccharide can undergo maturation in the Golgi resulting in a Man 8 -13 GlcNAc 2 carbohydrate structure, or it may be extended by an outer chain of variable size (up to 200 mannose residues) that is made up of a backbone of ␣1,6-mannosyl residues to which are attached branched ␣1,2and ␣1,3-mannosyl side chains.
Some of the enzymes involved in the elaboration of O-linked oligosaccharides and in the synthesis of N-linked outer chains have been identified, and their structural genes have been isolated. At least four different genes of the seven-membered PMT1-7 gene family encoding dolichyl phosphate-D-mannose: protein O-D-mannosyltransferases are responsible for initiating the O-linked glycans (4). Protein O-glycosylation is essential for cell function because mutants of S. cerevisiae lacking different combinations of three of the PMT genes are not viable (4). The OCH1 gene is responsible for adding the first ␣1,6mannose residue involved in initiating N-linked outer chain elaboration (5,6). Two enzymes in particular have been shown to participate in both O-and N-linked glycosylation. The KRE2/ MNT1 gene encodes a medial Golgi ␣1,2-mannosyltransferase required for the addition of the third mannose residue on O-linked chains (7,8) and is also implicated in N-linked outer chain oligosaccharide synthesis (9,10). The O-linked trisaccharide can be further extended by one or two ␣1,3-linked mannoses. The fourth mannose residue is added by the Golgi localized ␣1,3-mannosyltransferase Mnn1p, which also terminally mannosylates core and outer chain modified N-linked glycans (8,(11)(12)(13).
To further characterize protein glycosylation in yeast and identify some of the responsible glycosyltransferases, we have studied a gene family encoding proteins that are highly homologous to Kre2p/Mnt1p. This nine-membered KTR mannosyltransferase gene family contains the KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6, and KTR7 genes (14 -16). As with other known glycosyltransferases (17), all genes in this family are predicted, and some have been shown to encode type-II membrane proteins with a short cytoplasmic N terminus, a membrane-spanning region, and a highly conserved catalytic lumenal domain (8, 10, 14 -16). Similarity between different family members is variable (14 -16). For example, Yur1p and Ktr2p are most similar sharing 62% identity, followed by Ktr1p and Ktr3p with 54% identity. Ktr6p and Ktr5p with 24% identity constitute the two most divergent enzymes in the family. Initial functional characterization of Yur1p, Ktr1p, and Ktr2p has revealed that they are Golgi mannosyltransferases involved in N-linked glycosylation, possibly as redundant enzymes, but when inactivated they show no defects in O-linked glycosylation (9,10,14). Here, we report that KTR1, KTR3, and KRE2, which form a related subfamily encode ␣1,2-mannosyltransferases that together add the sec-ond and the third ␣1,2-linked mannose residues on O-linked carbohydrate chains and that also participate in N-linked outer chain elaboration.
Gene Disruptions-Deletional disruptions of KRE2 and KTR1 are described elsewhere (8,10). Disruption of the KTR3 locus was made by a single-step gene replacement procedure (19). First, the KTR3 gene was synthesized in vitro by the polymerase chain reaction (20) using the pfu DNA polymerase (Stratagene, La Jolla, CA) and CCACCACTT-TCAGCATGG and GAACCCAAGAAGGCACTAG as 5Ј and 3Ј primers, with yeast genomic DNA as a template. The 2-kilobase fragment obtained was then subcloned directly in the pTZ/PC vector as described previously (14). A 294-base pair EcoRV coding sequence fragment was removed and replaced by a 1.8-kilobase EcoRI fragment containing the complete HIS3 gene. A linear 3.5-kilobase NcoI fragment containing the complete HIS3 gene as well as the flanking regions plus the coding sequences of KTR3 gene was gel purified and integrated into the isogenic diploid SEY6210 strain. Integrants were verified by Southern analysis (data not shown).
Immunoblotting-The extracellular proteins Kre9p and invertase were expressed at high levels from a 2-based multicopy vector in yeast strains bearing different disruptions and concentrated from cultures growing exponentially in YNB selective medium containing 5% (v/v) glycerol and 2% glucose using Amicon centriprep concentrators (W. R. Grace & Co. Danvers, MA). Immunoblots were carried out as described (10, 21) using anti-Kre9p (22) and anti-invertase antisera (10), and detection was performed by an enhanced chemiluminescence procedure (Amersham, Oakville, ON, Canada).

Functional Characterization of Three Members of the KTR
Gene Family-The possible roles of members of the KTR family in O-and N-linked glycosylation remain to be determined. A testable assumption is that those proteins in the family that are most closely related are most likely to possess functional similarities. A relational homology tree constructed using the catalytic domains of each protein clarifies such relationships and has permitted an attempt to functionally group the different enzymes (16). One conclusion drawn from this analysis was that Kre2p is most related to Ktr1p and Ktr3p, indicating that these three proteins form a subfamily with possible related functions (16).
Recent in vitro studies using O-linked type substrates demonstrated that Ktr1p is a ␣1,2-mannosyltransferase with enzymatic properties highly similar to those of Kre2p, suggesting that these two enzymes act in similar ways (23). To explore these relationships and examine the role of Ktr3p and reassess the role of Ktr1p and Kre2p in the synthesis of O-linked chains, the extent of O-glycosylation in yeast strains bearing mutations in these genes was investigated. Deletional disruptions of KRE2 and KTR1 were previously obtained (8,10), and a disruption of the KTR3 locus was made (see "Experimental Procedures"). Double and triple disruptions were subsequently constructed using standard genetic techniques. The single and all double null mutants showed no growth defects when compared with the wild type strain. However, the haploid kre2 ktr1 ktr3 triple null mutant had a slow growth phenotype, indicating a genetic interaction between the three genes. 2 Ktr1p, Ktr3p, and Kre2p Participate in Adding the Second and Third ␣1,2-linked Mannose Residues on O-linked Oligosaccharides-O-glycosylation was examined in yeast strains bearing different disruptions by following the mobility of Kre9p (Fig. 1), a protein involved in cell wall assembly that is highly O-mannosylated but receives no N-linked modifications (22). Kre9p produced in a wild type strain migrates at an apparent mass of 55 kDa (10,21,22). As previously observed, Kre9p isolated from a kre2 null strain migrated more quickly than did the wild type Kre9p, with an apparent molecular mass of approximately 47 kDa (10,21), whereas Kre9p produced by ktr1 (10) or ktr3 2 single null disruptants was indistinguishable from that produced by a wild type strain. Kre9p produced in a ktr1 ktr3 double null mutant showed a small increase in mobility, whereas Kre9p produced in a kre2 ktr3 mutant was similar to Kre9p produced in a kre2 single mutant. However, a ktr1 mutant exacerbated the O-mannosylation defects of a kre2 mutant as the apparent molecular mass of Kre9p was approximately 5 kDa smaller (42 kDa) than when produced in a kre2 mutant alone. Kre9p was smallest (ϳ38 kDa) when produced in a triple ktr1 ktr3 kre2 mutant, indicating a cumulative involvement of all three proteins in O-linked modifications.
To determine the extent of this involvement, an analysis was made of total O-linked carbohydrate chains present on glycoproteins of mutants carrying different combinations of the disrupted genes (Fig. 2). O-linked carbohydrate chains were specifically released from the total glycoprotein fraction of in vivo [ 3 H]mannose-labeled yeast cells by ␤-elimination and resolved by chromatography (10, 14, 21; see "Experimental Procedures"). The wild type strain ( Fig. 2A) and the ktr1 and ktr3 single null mutants 2 showed the normal profile of five oligosaccharide peaks, as was the case with the ktr1 ktr3 double null mutant (Fig. 2B). As expected, the pattern obtained from the kre2 null strain gave two major peaks (Man1-Man2; Fig. 2C), consistent with failure to add the third ␣1,2-linked mannose residue and a minor peak indicating that a small proportion of total glycoproteins received a third mannose, an effect previously seen (7). Other yeast enzymes are, therefore, able to carry out this particular reaction to a limited extent.
A significant reduction in mannosylation is evident in both the ktr1 kre2 and ktr3 kre2 double null mutants, each of which gave two peaks (Man1-Man2) but with a decreased proportion of glycoproteins receiving 2 mannose residues when compared with a kre2 disruptant alone (Fig. 2, D and E), suggesting an involvement of both Ktr1p and Ktr3p in adding this mannose to O-modified chains. In those two double mutants, the proportion of O-glycoproteins receiving a third mannose is less than in a kre2 single null, suggesting that both Ktr1p and Ktr3p have a limited capacity to add a third mannose on O-linked chains. In a ktr1 ktr3 kre2 triple mutant, the predominant O-linked oligosaccharides assembled were composed of a single mannose (Fig. 2F), indicating that collectively Ktr1p, Ktr3p, and Kre2p are responsible for adding the third mannose and most of the second on O-linked glycans. At least one other enzyme, still unidentified but possibly encoded by a member of the KTR gene family (16), is responsible for the residual level of attachment of a second mannose (Fig. 2F). All the enzymes now known to be responsible for the assembly of yeast O-linked sugars are outlined in Scheme 1.
Ktr1p, Ktr3p, and Kre2p Jointly Participate in N-Glycosylation-The Kre2p mannosyltransferase is presumably involved in the elaboration of N-linked outer chain glycans (9, 10), but the precise mannoses added by this enzyme remain to be determined. In view of the cumulative role of KTR1, KTR3, and KRE2 in O-glycosylation, the effect on N-glycosylation of inactivating different combination of these genes was assessed. The carbohydrate chains of invertase, a specifically N-modified protein, were analyzed by measuring the mobility of the secreted form of this protein (Fig. 3), which is extensively modified with core oligosaccharides extended with outer chain glycans (24,25).
The extracellular wild type protein, as is the case when it is synthesized in ktr1 and ktr3 single null mutants, 2 has a molecular mass of around 150 kDa (Fig. 3). As seen before (10), invertase produced in a kre2 null mutant receives less Nmodification (ϳ143 kDa). The oligosaccharides attached to invertase synthesized in a ktr1 ktr3 double null mutant appear unaffected because the approximate molecular mass of the polypeptide chains made in this particular strain equals that of wild type proteins. The oligosaccharidic defects of invertase produced in a ktr1 kre2 double null mutant were similar to those observed in a kre2 single null mutant, whereas a disruption in KTR3 slightly exacerbates the N-mannosylation defects

SCHEME 1. Assembly of the S. cerevisiae O-linked oligosaccharide structures.
Arrows depict ␣1,2and ␣1,3-linkages between mannose residues. The various enzymes attaching the different mannose residues are indicated. The Pmtp family of protein O-mannosyltransferases adds the first mannose on the Ser/The residues in the ER (4). Ktr1p and Ktr3p along with the Kre2p/Mnt1p ␣1,2-mannosyltransferase participate in the addition of the second mannose residue onto O-linked chains. Kre2p has been known to be the main enzyme responsible for the addition of the third mannose on O-glycans (7,8). Ktr1p and Ktr3p are also able to add this particular mannose, although to a lesser extent than Kre2p (see Fig.  2). The Mnn1p ␣1,3-mannosyltransferase attaches the fourth mannose residue in the linear chain of up to five mannose residues (11)(12)(13). seen in a kre2 mutant because invertase synthesized in a ktr3 kre2 mutant has a molecular mass of ϳ137 kDa. However, invertase secreted from a ktr1 ktr3 kre2 triple null mutant was found to have a molecular mass of ϳ129 kDa demonstrating that, as was the case with O-glycosylation, all three proteins are collectively involved in N-linked modifications.

DISCUSSION
Ktr1p and Ktr3p are involved in the elaboration of O-linked glycans by adding the second mannose in the linear five mannose carbohydrate chain. This was not initially seen because yeast strains bearing ktr1 and ktr3 single or double disruptions possess a normal O-glycosylation pattern. Only in a ktr1 ktr3 kre2 triple mutant is the full effect of the absence of these enzymes apparent, where the third mannose is absent and a severely reduced level of mannose 2 is observed. In the absence of Ktr1p and Ktr3p, Kre2p is able to add both the second and third ␣1,2-mannose residue in the linear five mannose carbohydrate chains. Ktr1p and Ktr3p are to a limited extent also capable of attaching the third ␣1,2-mannose onto O-linked chains. All 3 enzymes, therefore, appear to have overlapping roles in the addition of both the second and third ␣1,2-linked mannose residues of O-glycosyl chains in yeast (see Scheme 1). The exact in vivo contribution of each enzyme to the synthesis of the second and third mannose linkages in wild type cells remains to be determined.
The results presented here demonstrate that Ktr1p, Ktr3p, and Kre2p are also implicated in N-linked outer chain elaboration. These transferases do not participate in N-linked core glycan synthesis because the molecular mass of the core modified oligosaccharide received by the late Golgi protein Kex1p (26) is the same in the ktr1 ktr3 kre2 triple null mutant and in a wild type strain. 2 Therefore, the marked reduction in the sugar chains received by invertase in the ktr1 ktr3 kre2 triple mutant indicates that Ktr1p, Ktr3p, and Kre2p act in the Golgi apparatus to elaborate outer chain glycans of N-linked oligosaccharides by making some of the branched mannosyl side chains that are attached to the ␣1,6-mannosyl residues backbone (see Fig. 3). Of the five mannoses constituting this backbone sugar chain, all are substituted by at least one ␣1,2-linked mannose residue, and three are modified by at least two ␣1,2linked mannoses. Because the structure of these N-linked branched mannosyl side chains is reminiscent of O-linked oligosaccharides, it is a reasonable speculation that Ktr1p, Ktr3p, and Kre2p collectively participate in establishing some of these outer chain ␣1,2-linkages. Recent in vitro enzymatic studies are consistent with this (23). Both Kre2p and Ktr1p utilize the N-glycan type oligosaccharidic substrate, Man [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] GlcNAc, which contains the ␣1,6-mannose outer chain backbone attached to the core sugar but lacks the ␣1,2-mannose containing branches (see Fig. 3), indicating that both enzymes have the ability to add a first ␣1,2-linked mannose residues to the outer chain backbone. Moreover, the mannosyltransferase reaction involving Kre2p exhibited biphasic kinetics, with an increase in mannose incorporation into product during the second part of the reaction. Such an activity is in agreement with this enzyme sequentially adding two mannose residues and parallels its role in O-mannosylation.
The basis for the multiplicity of mannosyltransferases and the complexity of their involvement in the synthesis of O-and N-mannosyl chains can be addressed in the context of redundancy in gene families. The members of the mannosyltransferase family discussed here have related and overlapping functions that can take at least two forms. Firstly, multiple enzymes act at single biosynthetic steps. For example, Kre2p, Ktr1p, and Ktr3p add the second O-linked mannose residue. Individual enzymes can also participate in more than one bio-synthetic step. Kre2p, Ktr1p, and Ktr3p, which can add the second mannose residue to O-linked chains, are also involved together in adding a third mannose residue, though to differing extents (see Scheme 1.).
The level of apparent redundancy observed between the enzymes can be explained in several ways. It is possible that each enzyme possesses a high affinity for a limited set of amino acid sequence contexts around the Ser/Thr and Asn residues at which mannosylation occurs. By having a number of enzymes of varying specificity, the cell is able to extend the range of mannosylated residues on glycoproteins. There is strong evidence for this notion with the first enzyme required in the O-mannosylation pathway, the protein mannosyltransferase, where some of the seven PMT encoded enzymes have been shown to have differing specificities for the mannosylation of peptide substrates of different sequence (4,21,27). Although there is no direct evidence for this conjecture in the KTR family, variable sequence specificities can be hinted at when comparing Kre9p O-mannosylation patterns with those seen through ␤-elimination in the total O-mannoprotein fractions of the kre2 ktr1 and kre2 ktr3 double null mutants. The mannosylation deficiencies of Kre9p are smallest in a kre2 ktr3 mutant, whereas the defects of the bulk of O-modified proteins are far more pronounced in this strain (see Figs. 1 and 2, D and E). This is consistent with these enzymes having different specificities with Kre9p sites being atypical.
The redundancy observed here can also be explained by a sequential action through intracellular compartmentalization. If related enzymes have broadly similar or overlapping substrate affinities but are in a distinct Golgi compartments, then those mannosylation sites on proteins that fail to be mannosylated in one compartment have subsequent glycosylation opportunities as they move through the later mannosyltransferase-containing cisternae. If this were the case, Ktr1p and Ktr3p that have less ability to add the third mannose in Oglycosylation might be found in cisternae before Kre2p, the enzyme most responsible for Man3 addition. This would build a level of redundant function into the processive extension of mannose chains in successive Golgi compartments and increase O-mannosylation efficiency. The Golgi targeting regions of Kre2p, Ktr1p, and Ktr3p are unrelated, consistent with them having different intracellular locations (8,10,16).
The addition of O-and N-mannose oligosaccharides in S. cerevisiae requires the action of many related mannosyltransferases. Studying the roles of gene families such as KTR will be informative both for the analysis of protein glycosylation and to offer insights into the biological reasons that allow such diversity of related gene products to occur.