(Received for publication, October 27, 1995; and in revised form, December 28, 1995)
From the
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 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
mannosyltransferases 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.
The covalent addition of glycans to secretory and membrane
proteins constitutes one of the major post-translational modifications
known to occur in eukaryotes. The biosynthetic pathway leading to N-glycosylation has been studied in considerable detail and
involves the ordered assembly of a core oligosaccharide on the lipid
carrier dolichol phosphate, which is embedded in the ER ()membrane. Once this oligosaccharide has been completed, it
is transferred onto specific asparagine residues of proteins and
subsequently altered by specific glycosidases and glycosyltransferases.
The elaboration and initial processing of N-linked
oligosaccharides in the ER are similar in all eukaryotes, but
subsequent phases of glycosylation are different in a broad range of
organisms (Tanner and Lehle, 1987; Herscovics and Orlean, 1993; Knauer
and Lehle, 1994; Lehle and Tanner, 1995).
In the yeast Saccharomyces cerevisiae, the N-linked core
oligosaccharide is mainly constituted of ManGlcNAc
and may undergo Golgi maturation resulting in
Man
GlcNAc
. In other cases,
glycoproteins traversing the Golgi have their core oligosaccharide
extended by outer chains containing up to 200 mannose residues (Ballou,
1990; Herscovics and Orlean, 1993; Lehle and Tanner, 1995). Protein N-glycosylation appears essential for cell function since
mutants of S. cerevisiae lacking protein subunits of the core
oligosaccharyltransferase or mutants defective in the synthesis of the
dolichol pyrophosphate-oligosaccharyl precursor are not viable
(Huffaker and Robbins, 1982; te Heesen et al., 1992, 1993;
Stagljar et al., 1994; Kelleher and Gilmore, 1994), although
the biochemical basis of this lethality remains unclear (Tanner and
Lehle, 1987; Lehle and Tanner, 1995).
The structure and biosynthesis of O-linked carbohydrate chains attached to serine and threonine show considerable evolutionary diversity. The primary reaction in the modification of mammalian O-linked proteins involves the attachment of a GalNAc that has been transferred from UDP-GalNAc within the Golgi (Roth, 1984). The carbohydrate chains of mammalian O-linked modified proteins are variable in length and composition and include galactose, sialic acid, fucose, GalNAc, and GlcNAc (Elhammer and Kornfeld, 1984; Roussel et al., 1988; Jentoft, 1990; Krijnse Locker et al., 1992). In contrast, it has been demonstrated that in S. cerevisiae O-modified proteins possess a linear carbohydrate chain consisting of up to 5 mannose residues (Tanner and Lehle, 1987; Herscovics and Orlean, 1993; Lehle and Tanner, 1995).
Some of the structural genes coding for
yeast mannosyltransferases have been isolated. OCH1 encodes
the first 1,6-mannosyltransferase involved in initiating outer
chain elaboration (Nakayama et al., 1992; Nakanishi-Shindo et al., 1993). KRE2/MNT1 is the only known
1,2-mannosyltransferase gene isolated to date
(Häusler and Robbins, 1992) and is implicated in N-linked outer chain oligosaccharide synthesis (Hill et
al., 1992) and is also responsible for the addition of the third
mannose residue of O-linked carbohydrate chains
(Häusler et al., 1992). Outer chain and
core modified oligosaccharides are brought to completion by the action
of a terminal
1,3-mannosyltransferase encoded by the MNN1 gene and similarly to Kre2p/Mnt1p, Mnn1p also mannosylates O-linked glycans (Ballou, 1990; Yip et al., 1994).
To examine how post-translational modifications occur in Saccharomyces cerevisiae and to further define the responsible enzymes, we have functionally characterized three members of the KRE2/MNT1 putative mannosyltransferase gene family. This growing gene family was known to contain KRE2/MNT1, YUR1, KTR1, KTR2 (Häusler and Robbins, 1992; Lussier et al., 1993), and recently two other homologues, KTR3 and KTR4, have been found by the yeast genome project (Mallet et al., 1994). These genes are predicted to encode type II membrane proteins with a short cytoplasmic N terminus, a membrane-spanning region, and a highly conserved catalytic lumenal domain. While the precise role of Kre2p/Mnt1p as a mannosyltransferase in O-glycosylation has been established (Häusler and Robbins, 1992; Häusler et al., 1992), the role of the other genes remains to be determined. We have carried out a functional analysis of Yur1p, Ktr1p, and Ktr2p and demonstrate that they are mannosyltransferases involved in N-linked glycosylation.
Figure 6:
Mannosyltransferase activity in vitro. Total membrane preparations from a kre2 ktr1 ktr2 yur1 quadruple null strain (SEY 6210) overexpressing each gene
individually from YEp351 (Hill et al., 1986) were assayed for
their ability to transfer of [C]mannose from
GDP-[
C] mannose to a specific acceptor. Assays
were carried out as described under ``Experimental
Procedures.'' Values are given as specific activities obtained
with
-methylmannoside or mannoprotein prepared from the quadruple
null strain as acceptors.
Figure 1: Topologic representation and sequence similarities of Kre2p Ktr1p, Ktr2p, and Yur1p. A, the different members of the family are presumed to be oriented as a type II membrane-anchored protein, a topology characteristic of all isolated glycosyltransferases that consists of a short N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, and a large C-terminal lumenal catalytic domain (Shaper and Shaper, 1992; Kleene and Berger, 1993). The catalytic domain is linked to the transmembrane domain by a ``stem'' region thought to be devoid of secondary structure. B, the degree of sequence homologies between the different proteins is represented as percentage of identities over the smallest protein and was calculated from sequence alignments with gaps to maximize homology.
In view of their sequence and structural
similarities with Kre2p, the possible role of the KTR1, KTR2 and YUR1 gene products as protein
mannosyltransferases was analyzed. One-step gene replacements were
carried out using different marker genes (see ``Experimental
Procedures'' and Fig. 2). KRE2 and KTR2 single gene disruptions were previously shown to have no growth
phenotypes at 30 °C (Häusler et al.,
1992; Lussier et al., 1993). Analysis of spore progeny derived
from SEY6210 ktr1::LYS2 or yur1::HIS3 heterozygotes
showed that neither gene was essential for cell viability nor were they
required for normal vegetative cell growth. ()To assess
whether a haploid strain carrying deletions in several of these genes
possessed a more severe phenotype, double, triple, and quadruple
disruptions were sequentially constructed using standard genetic
techniques. Meiotic tetrads segregating combinations of these disrupted
genes were dissected and haploid spore progeny grown at 30 °C.
Haploid strains harboring ktr1:LYS2 ktr2::URA3 yur1::HIS3 triple null mutations or haploids carrying a set of four
disruptions were viable and did not grow noticeably slower than wild
type cells.
Figure 2: Disruptions of the KRE2, KTR1, KTR2 and YUR1 genes. Restriction sites and construction of the different disruptions are shown. Black boxes represent DNA fragments. The open reading frames corresponding to each genes are indicated. Each gene was disrupted by a particular auxotrophic marker gene. For details, see ``Experimental Procedures.''
Figure 3:
-Elimination profiles. Paper
chromatograms of manno-oligosaccharides released by
-elimination
from bulk yeast glycoproteins of wild type cells (SEY 6210), and of the
same strain where KRE2 or KTR1, KTR2, and YUR1 were disrupted. Aliquots of extracts corresponding to
equal amounts of cells were run on thin-layer plates (also see
``Experimental Procedures''). The peaks designated M1-M5 represent carbohydrate chains bearing one to five
mannoses. M1, M2, and M3 co-migrate with
mannose, maltose, and raffinose standards.
The extent of O-glycosylation in yeast strains with mutations in these genes was also analyzed by measuring the mobility of a yeast O-glycoprotein, Kre9p. Kre9p is an extracellular matrix protein involved in cell wall assembly that is extensively O-mannosylated but lacks N-linked modifications (Brown et al., 1993). When synthesized in a wild type strain, Kre9p migrates at an apparent mass of 55 kDa. As expected, 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 (Fig. 4). However, Kre9p produced by ktr1, ktr2, or yur1 single null disruptants or by a triple null mutant strain was indistinguishable from that produced by a wild type strain.
Figure 4:
Immunological detection of Kre9p
synthesized in wild type, kre2, yur1, ktr1, ktr2, and yur1 ktr1 ktr2 triple null mutants. Kre9p
was overexpressed from plasmid YEp351 (Hill et al., 1986) in
different yeast strains and concentrated from exponentially growing
cultures (Brown et al., 1993). Yeast extracellular protein
extracts were immunoblotted with affinity-purified anti-Kre9p
polyclonal antibodies (see ``Experimental Procedures''). The
molecular mass standards are shown in kilodaltons. The S.
cerevisiae O-linked oligosaccharide structures are also shown. Arrows depict 1,2, and
1,3 linkages between
mannoses. The Kre2p/Mnt1p
1,2-mannosyltransferase is responsible
for the addition of the third mannose in a medial Golgi compartment
(Ballou, 1990; Häusler et al., 1992;
Lussier et al., 1995b).
Figure 5:
Immunological detection of invertase
synthesized in wild type and different null mutants. Invertase was
overexpressed from plasmid YEp351 (Hill et al., 1986) in
different yeast strains and concentrated from exponentially growing
cultures. Yeast extracellular protein extracts were immunoblotted with
anti-invertase polyclonal antibodies (see ``Experimental
Procedures''). The molecular mass standards are shown in
kilodaltons. The S. cerevisiae possible N-linked
oligosaccharide structures are also shown (adapted from Ballou(1990)).
1,4,
1,6,
1,2, and
1,3 linkages between mannoses of
the core and outer chain are depicted. x equals 10 on
average.
As found previously (Hill et
al., 1992), invertase synthesized in a kre2 null mutant
has a molecular mass ( 137 kDa) that is smaller than the secreted
wild type protein (
145 kDa). In contrast, the carbohydrate chains
of invertase produced in ktr1, ktr2, or yur1 single null mutants appear to be intact, as the molecular mass of
the protein made in these strains is wild type (
145 kDa).
Similarly, in ktr1 ktr2, ktr1 yur1, or ktr2 yur1 double null mutants, no obvious reduction in size of invertase was
apparent. However, invertase synthesized in a ktr1 ktr2 yur1 triple null mutant possessed a molecular mass of approximately
127 kDa. Invertase was smallest (
120 kDa) when produced in a
quadruple kre2 ktr1 ktr2 yur1 mutant, indicating a cumulative
involvement of all four proteins in N-linked modifications.
Figure 7:
Killer toxin sensitivity phenotypes of
wild type and different null mutants. Concentrated K1 killer toxin was
spotted on a lawn of approximately 1 10
/ml cells
from a fresh culture of each strain (see ``Experimental
Procedures''). After subsequent incubation, toxin sensitive cells
show a killing zone in the growth lawn. Toxin-resistant cells grow in
the presence of the toxin and show no killing
zone.
Yeast strains harboring single and double mutations, as well as a triple null mutation, of KTR1, KTR2 and YUR1 were assayed for killer toxin sensitivity (Fig. 7). When evaluated by seeded plate assays, the wild type toxin-sensitive SEY6210 strain displayed a large killing zone (15 mm), whereas the kre2 mutant was completely toxin-resistant. A strain bearing a single KTR1 disruption showed no phenotypic resistance to K1 killer toxin. When compared to the wild type strain, yur1 (10 mm) and ktr2 (12.5 mm) single null disruptions were both partially resistant to the killer toxin, yur1 being more resistant. Yeast cells carrying ktr1 ktr2, ktr1 yur1, or ktr2 yur1 double disruptions all displayed pronounced levels of resistance. The ktr1 ktr2 (10 mm) double null strain showed a stronger phenotype than either the ktr1 or ktr2 single null. Both ktr1 yur1 (8 mm; clear) and ktr2 yur1 (7 mm; fuzzy) double nulls are more resistant than a strain carrying a yur1 null mutation indicating that disruption of either KTR1 or KTR2 exacerbates the cell wall defect of a yur1 mutant. Finally, a ktr1 ktr2 yur1 triple null mutant is almost totally resistant, suggesting a cumulative effect on the reduction of carbohydrate chains leading to killer resistance. These results thus appear to also implicate N-linked chains as part of the killer receptor.
The killer phenotype of some single null mutants allowed a
test of possible suppression of the loss of one gene by another
homologous counterpart. Ktr1p, Ktr2p, and Yur1p could not suppress the
killer resistance of a KRE2 null mutant and thus could not
functionally substitute for it. Functional suppression
could only be established between the YUR1 and KTR2 genes, with overexpression of KTR2 in a strain carrying a yur1 null mutation completely suppressing the yur1 killer resistance phenotype, indicating that when expressed at
very high levels, Ktr2p has the capacity to substitute in vivo for the absence of the Yur1p.
These two proteins are
62% identical and constitute the most homologous pair among members of
the Kre2p family (Lussier et al., 1993; Mallet et
al., 1994).
Figure 8: Cellular localization of Ktr1p, Ktr2p and Yur1p by indirect immunofluorescence. Diploid yeast (SEY6210) containing the KTR1 gene or epitope-tagged KTR2 or YUR1 on multicopy plasmid, YEp352 (Hill et al., 1986), were fixed, spheroplasted, attached to polylysine-coated glass slides, and then incubated with affinity-purified anti-Ktr1p Ab or 12CA5 monoclonal antibody and DAPI. Texas Red-coupled secondary Ab was added to detect antigen-immunoglobulin complexes. Cellular DAPI staining of nuclear and mitochondrial DNA is shown.
Kre2p is an 1,2-mannosyltransferase
(Häusler and Robbins, 1992;
Häusler et al., 1992), and we present here
evidence showing that Ktr1p, Ktr2p, and Yur1p are also involved in
protein glycosylation. As Kre2p is a mannosyltransferase adding the
third mannose residue on O-linked mannose carbohydrate chains,
the possible role of Ktr1p, Ktr2p, and Yur1p in O-glycosylation was analyzed. Experiments indicated that
neither the O-glycosylation of total yeast mannoprotein nor
the O-glycosylation of Kre9p is affected by these proteins,
since no differences from wild type were seen in single or triple null
mutants (ktr1 ktr2 yur1).
The influence of KTR1,
KTR2, and YUR1 gene disruptions on protein N-glycosylation was analyzed. The N-glycosylated
protein invertase was found to be underglycosylated in the ktr1
ktr2 yur1 triple null mutant compared to a wild type strain but
not in single or double disruptants, except in the case of kre2 where, as expected, an effect was seen (Hill et al.,
1992). Invertase receives even less glycosylation when synthesized in
the quadruple, ktr1 ktr2 yur1 kre2, null strain but is still
heavily N-modified since its migration pattern (120 kDa)
remains considerably larger than the molecular mass of the protein
predicted from the DNA sequence (59 kDa). These results are consistent
with these enzymes having redundant functions in N-linked
glycosylation.
Possible additional roles for Ktr1p, Ktr2p, Yur1p,
and Kre2p were also assessed. S. cerevisiae carries several
phosphoinositol)-containing sphingolipids, specifically inositol
phosphoceramides, mannosylinositol phosphoceramides (which contain a
mannose attached to the inositol), and mannosyl(inositol phospho) ceramides (which is substituted with one mannose and 2
phosphoinositol groups). Ktr1p, Ktr2p, Yur1p, and Kre2p do not appear
to be involved in this lipid mannosylation, as none of the strains
containing single or multiple deletions of these genes lacked any of
the mannosylated inositol phospho-ceramides. (
)Another
possibility is that Ktr1p, Ktr2p, Yur1p, and Kre2p elaborate part of
the short
-linked mannose side chains found on protein-bound GPI
anchors. None of the enzymes could be solely responsible for a single
biosynthetic step in GPI core synthesis, since this is an essential
process in yeast (Leidich et al., 1994, 1995), and none have a
lethal phenotype when disrupted. Strains carrying single or multiple
deletions of these four genes all synthesized normal GPI anchors (Sipos et al., 1995).
Evidence for the function of Yur1p, Ktr1p
and Ktr2p as mannosyltransferases was obtained by evaluating their in vitro enzymatic activities. Using -methylmannoside and
oligosaccharides found on mannoproteins from a kre2 ktr1 ktr2 yur1 quadruple null strain as acceptors, YUR1 overproducing
cells showed a 5.5- and 2.1-fold increase in activity over background
respectively. These values are similar to those obtained by
overproduction of the known mannosyltransferase encoding gene, KRE2. By comparison, when
-methylmannoside was used as a
saccharide acceptor, high levels of expression of KTR1 and KTR2 did not result in activity levels higher than those
obtained with the parental quadruple null strain. However, when the
mannoprotein fraction was used as an acceptor, increased levels of
activity similar to those obtained with YUR1 were reproducibly
found with both KTR1 and KTR2, indicating that their
gene products also are mannosyltransferases. The activity difference
seen between these enzymes in the two assays suggests that these
mannosyltransferases differ in substrate specificity.
Further evidence that Ktr1p, Ktr2p, and Yur1p are mannosyltransferases comes from an in vivo analysis of their function. Strains carrying non-functional copies of KTR1, KTR2, and/or YUR1 genes, became to varying extents K1 killer toxin-resistant, the triple null mutant being most resistant. These results indicate that, as is the case with KRE2 null mutations, singly or in combination disruptions of KTR1, KTR2, and YUR1 lead to a reduced amount of N-linked glycans on cell wall mannoproteins perturbing the cell surface toxin receptor and leading to resistance. The fact that functional replacement by overproduction could only be obtained between the most similar gene pair, YUR1 and KTR2, also suggests that these mannosyltransferases likely perform different functions.
From the above results, it can
be concluded that Ktr1p, Ktr2p, and Yur1p are implicated as
mannosyltransferases in N-linked glycan elaboration. However,
these enzymes do not participate in the synthesis of the basic N-linked core oligosaccharide, as they are situated in the
Golgi apparatus and the core oligosaccharide is elaborated and
transferred to protein in the ER (Herscovics and Orlean, 1993; Lehle
and Tanner, 1995). Similarly, Ktr1p, Ktr2p, and Yur1p do not
participate in core Golgi modifications (see Fig. 5), as the
size of the core modified oligosaccharide received by the late Golgi
protein Kex1p (Cooper and Bussey, 1992) is the same in the triple ktr1 ktr2 yur1 null mutant and in wild type. Thus,
it is likely that the role of Ktr1p, Ktr2p, and Yur1p is to participate
in the elaboration of the outer chain glycans of N-linked
oligosaccharides. A number of distinct
1,2-linked mannosylation
reactions are required for the synthesis of the outer chain of yeast N-linked modified proteins (see Fig. 5), and we
speculate that Ktr1p, Ktr2p, Yur1p, and also Kre2p (Hill et
al., 1992) are partially responsible for establishing some of
these
1,2-linkages in the Golgi apparatus.
The cumulative
effect of multiple gene disruptions seen on the size of the N-linked carbohydrates carried by invertase and on the degree
of in vivo killer toxin resistance can be rationalized in
distinct ways that are not necessarily mutually exclusive. Ktr1p,
Ktr2p, Yur1p, Kre2p, and other similar mannosyltransferases could
function redundantly in the sense of having overlapping specificities.
Different forms of functional redundancy can be envisaged in the
context of a large family of glycosyltransferases elaborating complex
glycans. 1) More than one enzyme could be able to establish one
specific class of glycosyl linkage. This could happen in normal
vegetative growth or could be achieved by differential regulation under
specific conditions. The PMT gene family encoding protein O-mannosyltransferases constitutes an example of this type of
redundancy (Strahl-Bolsinger et al., 1993; Lussier et
al., 1995a; Immervoll et al., 1995). 2) Conversely, an
individual mannosyltransferase may catalyze the assembly of one type of
carbohydrate linkage to more than one type of oligosaccharide, as is
the case for the Mnn1p terminal 1,3-mannosyltransferase (Ballou,
1990; Cooper and Bussey, 1992; Graham et al., 1994; Yip et
al., 1994; Lussier et al., 1995b). 3) Added complexity
may arise if members of the Kre2p family are not localized in the same
Golgi compartment. Some member proteins could possess the same
enzymatic specificity, but their Golgi retention signal would be
different, targeting them to distinct intracellular locations. A key
element in the targeting of Kre2p to the medial Golgi has been shown to
lie in the N-terminal region (Lussier et al., 1995b). This
interesting region of the sequence of Kre2p, Ktr1p, Ktr2p, and Yur1p is
unique to each protein hinting that they may be localized to different
Golgi subcompartments.
Taken together, our results indicate that Ktr1p, Ktr2p, Yur1p, and Kre2p are involved in the elaboration of outer chain N-linked glycans. The specificity of the mannosyltransferase reactions catalyzed by these four enzymes is likely to vary, as each showed a different pattern of activity toward the two acceptors used in our in vitro assays. Multicopy suppression of the phenotype caused by one deleted transferase by another provides an indication of at least some partial overlap of mannosyltransferase specificity. The lack of multicopy suppression, however, provides little information as enzymes with similar specificity may reside in different Golgi compartments or be differentially regulated. A clearer picture of the overall specificity, location, and regulation of the KRE2/MNT1 family awaits the identification and characterization of the entire gene family in S. cerevisiae, a goal likely attainable by the completion of the genome sequence of this organism.