From the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom
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ABSTRACT |
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In the yeast Saccharomyces cerevisiae
many of the N-linked glycans on cell wall and periplasmic
proteins are modified by the addition of mannan, a large
mannose-containing polysaccharide. Mannan comprises a backbone of
approximately 50 The glycoproteins of the cell wall of the yeast
Saccharomyces cerevisiae are modified with both
N-linked and O-linked glycans. The
O-linked structures are chains of 4-5 mannoses attached to serines and threonines, whereas the N-linked glycans
comprise the conventional core structure to which can be attached a
long outer chain structure of up to 200 mannose residues (1, 2). This
"mannan" modification is so extensive that the mannoproteins contribute up to 40% of the dry weight of the yeast cell wall (3).
Mannans provide an external layer to the wall, which is believed both
to contribute to its structural integrity and serve to exclude
hydrolytic enzymes. Analysis of the structure of the S. cerevisiae mannan shows that it consists of a long The synthesis of the mannan structure in S. cerevisiae has
been extensively investigated by both genetic and biochemical means. The mannan outer chains are only attached to the N-linked
glycans of proteins destined for inclusion in the cell wall or
residence in the periplasmic space. The synthesis begins in the Golgi
apparatus where the first Studies on mannan synthesis in S. cerevisiae are facilitated
by mannan not being essential for viability. Survival without mannan
is, however, dependent on cells being able to sense the cell wall
defect, presumably to allow up-regulation of other cell wall components
especially chitin (19). Thus mutations that result in defective mannans
show synthetic lethality with components of the protein kinase C
pathway, which regulates the expression of cell wall components (15,
20, 21). Cell wall integrity is likely to be particularly critical
during budding, and again survival without mannan seems also to depend
both on the actin cytoskeleton being well organized and on a functional
mitotic check point, which allows bud formation to be slowed (22-24).
In this study, we investigate a Golgi membrane protein of previously unknown function that was initially identified as corresponding to a
bud emergence delay mutation bed1 (22). It was subsequently shown to correspond also to a previously identified mannan synthesis mutant mnn10 (25, 26). Because Mnn10p is distantly related to Mnn11p, one of the components of the Anp1p-containing complex, we
investigated its relationship to this complex. We show here that it is
a further component of the complex that seems to comprise five
proteins. This large structure does not appear to contain further
proteins. Investigation of the activity of the complex in wild type and
mutant strains indicates that the Mnn10p and Mnn11p proteins are
responsible for the majority of the Yeast Strains and
Plasmids--
S. cerevisiae strain SEY6210
(Mat Immunoblotting and Immunoprecipitation--
Proteins separated
by SDS-polyacrylamide gel electrophoresis were electrophoresed onto
nitrocellulose and probed with antibodies in phosphate-buffered saline,
2% dried milk, 0.5% Tween 20. Immunoblotting and immunoprecipitations
were performed with monoclonals against the Myc epitope (9E10), the HA
epitope (12CA5), or ribosomal protein Tcm1p (kindly provided by Jon
Warner) or with rabbit antisera against both tags (Santa Cruz
Biotechnology) or Anp1p (12). Peroxidase-conjugated anti-rabbit and
anti-mouse secondary antibodies were detected by chemiluminescence
(ECL, Amersham Pharmacia Biotech). Total protein samples were
prepared by resuspending log-phase yeast at one
A600 unit per 20 µl of SDS buffer, bead
beating for 5 min at 4 °C (425-600-µm glass beads, Sigma), and
denaturing at 65 °C for 5 min. Immunoprecipitates were prepared from
unlabeled cells as described previously (15). Precipitation of protein A fusions was performed essentially as described previously but adapted
for metabolically labeled cells. Thus four A600
units of log-phase cells were labeled with 200 µCi of
Tran35S-label (1,170 Ci/mmol, ICN) in 0.5 ml for 30 min at
30 °C, spheroplasted as for the unlabeled cells, and resuspended in
0.5 ml of T/D/I buffer (10 mM triethanolamine, pH 7.5, 150 mM NaCl, 1% digitonin, 2 mM EDTA supplemented
by protease inhibitors). After centrifugation at 14,000 × g for 10 min to remove cell debris, the supernatant was
added to 15 µl of IgG-Sepharose (Amersham Pharmacia Biotech), rocked
gently overnight, and the beads washed five times for 1 h in 0.4 ml T/D/I, before cleavage overnight in 20 µl of T/D/I with 5 units of
TEV protease (Life Technologies, Inc.), all steps after spheroplasting
being at 4 °C. The supernatant from the beads was removed, divided
in two and treated with, or without, 500 units of endoglycosidase H
(Endo H) according to the manufacturer's instructions (New England
Biolabs). Protein was precipitated by methanol/chloroform extraction,
resuspended in SDS sample buffer, separated by SDS-polyacrylamide gel
electrophoresis, and the gel was treated with Amplify (Amersham
Pharmacia Biotech) and fluorographed.
Gel Filtration--
Protein complexes prepared by IgG-Sepharose
precipitation and TEV protease cleavage from 1,000 A600 units of cells, or 14,000 × g supernatants from spheroplasts lysed in T/D/I buffer, were fractionated on a 34 × 1.5-cm column of Sephacryl S400 (Amersham Pharmacia Biotech). The column was run in 10 mM
triethanolamine, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 0.4% digitonin at 15 ml/h and 1 ml of fractions were collected.
100 µl of fractions were precipitated by methanol/chloroform
extraction with soybean trypsin inhibitor as a carrier, resuspended in
SDS buffer, and analyzed by immunoblotting. For standards,
thyroglobulin, catalase, and ferritin (Amersham Pharmacia Biotech) were
run on the same column under identical conditions and analyzed by
SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.
Mannosyltransferase Assays--
Protein A fusions or
Anp1p-containing complexes were isolated and used on the beads for
mannosyltransferase reactions as described previously (12, 15). The
reaction products were fractionated by gel filtration on a 14 × 1.0-cm Sephadex G10 column (Amersham Pharmacia Biotech) run in
deionized water. The radioactive peak fractions were pooled,
concentrated by lyophilizing, treated with mannosidases, and the
products reacted with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)
for separation by electrophoresis as described previously (12, 30) with
the reaction products from 50 to 100 A600 units of cells loaded per lane. The resulting gels were exposed to a PhosphorImager screen for 3 days (or 2 weeks for the
Mnn10p Is a Component of the Anp1p-Mnn9p Complex of the cis
Golgi--
To determine whether Mnn10p was a component of one of the
two Mnn9p-containing complexes, the same co-immunoprecipitation approach was followed that was originally used to reveal the
interactions between Mnn9p and the other members of the complexes (12).
Thus a triple HA-epitope tag was inserted at the end of the
MNN10 open reading frame by homologous recombination.
Protein blotting revealed that the tagged protein migrated as a single
band with an apparent molecular mass of 46 kDa, reasonably close to
that predicted from the sequence of the protein and the epitope tag (49 kDa, Fig. 1A). An anti-HA
monoclonal antibody was used to precipitate the protein from yeast
solubilized with the mild detergent digitonin. The immunoprecipitates
were then blotted with antibodies to Anp1p and Van1p, and Fig.
1B shows that Mnn10p is associated with Anp1p and not with
Van1p. Moreover, blotting of the supernatant following precipitation
revealed that the Anp1p was quantitatively precipitated by the tagged
Mnn10p, indicating that all of the Anp1p is associated with complexes
that contain Mnn10p (Fig. 1B).
Organization of the Anp1p-Mnn9p Complex--
Our previous analysis
of the Mnn9p-containing complexes had shown that Anp1p is associated
not only with Mnn9p but also with two other membrane proteins, Hoc1p
and Mnn11p. As with Mnn10p, these proteins were not found associated
with the Van1p-Mnn9p complex. This raises the question of how these
five proteins are organized. In each case it is known that the protein
associates with Anp1p. One possibility is that all five proteins are in
a single complex. Alternatively there could be distinct complexes containing Mnn9p-Anp1p and one or two of the other components. We
addressed this issue in two ways. First, we constructed strains with
different epitope tags at the C terminus of pairs of the proteins.
Immunoprecipitates were then performed using one anti-tag antibody, and
the precipitated proteins probed on a blot for the second tagged
protein. A representative experiment is shown in Fig.
2A for strains expressing
Mnn10p and Mnn11p, tagged with Myc and HA in both combinations. In both
cases, not only could the HA-tagged member of the pair be precipitated
with the anti-HA monoclonal antibody as expected, but it could also be
precipitated with the anti-Myc monoclonal antibody. This indicates that
Mnn10p and Mnn11p are present in the same complex. Likewise the
HOC1 open reading frame was tagged with the Myc epitope in
strains in which either Mnn10p or Mnn11p were tagged with HA. In both cases the Myc-tagged Hoc1p could be precipitated with antibodies to the
Myc or the HA tags, and also with a rabbit polyclonal against Anp1p.
Blotting of the digitonin lysate of the Hoc1p-Myc-expressing cells
revealed two bands, of which only the upper is present in blots of
cellular proteins solubilized directly in SDS-containing sample buffer
(Fig. 2B and data not shown). Because the tag is at the C
terminus, this suggests that proteases in the digitonin lysate remove
about 5 kDa from the NH2 terminus of the protein. Such
clipping in the region after the amino-terminal transmembrane domain
has been seen for many Golgi enzymes in both yeast and mammals and is
proposed to reflect this part of the enzymes being a flexible stalk
region (31, 32). Interestingly, this clipped form does not
co-precipitate with either Mnn10p, Mnn11p or Anp1p, which may reflect
removal by the protease of a region required for association in the
complex (Fig. 2B).
The only exception to this co-precipitation of the various
Anp1p-associated proteins was observed when tagged Hoc1p was
precipitated from a strain also expressing tagged Mnn10p. Although
precipitation of HA-tagged Mnn10p with anti-HA resulted in the
co-precipitation of Myc-tagged Hoc1p, only low levels of Mnn10p-HA were
precipitated when the precipitation was performed the other way round
with anti-Myc (Fig. 2B). One possibility is that the
C-terminal tag on Hoc1p lies close to the interface between Hoc1p and
Mnn10p, and that binding of the anti-Myc monoclonal antibody disrupts the interaction.
Precipitation of the Anp1p-containing Complex from Metabolically
Labeled Cells--
To investigate further the composition of the
Anp1p-containing complex and examine the possibility that it contains
yet further components, a second approach was used to address the
composition of the complex. For this a protein A tag was attached to
the C terminus of each of the components of the complex in separate strains. The spacer between the protein and the tag contained a
cleavage site for the sequence-specific protease from TEV. This allows
the tagged proteins to be captured on IgG-Sepharose, and then
proteolytically eluted under native conditions with minimal background
(15). The isolation was initially performed with strains expressing
either protein A-tagged Anp1p or tagged Van1p, or no tagged protein.
Fig. 3A shows that the two
tagged proteins precipitate a distinct pattern of bands, consistent
with their being in two distinct complexes that share only the
component Mnn9p. This analysis was then extended to the other
components of the Anp1p-containing complex, and Fig. 3B
shows the results of such a precipitation and elution performed on
35S-labeled strains in which the different components of
the complex had been tagged. A thirteen residue spacer including the
TEV cleavage site is left on the protein following protease treatment,
and this results in a small reduction of mobility compared with the nontagged version. In most cases all the other proteins can be identified as co-precipitating with a particular component. Analysis of
the gels is complicated by the fact that Mnn10p and Hoc1p apparently co-migrate (consistent with their predicted molecular weights differing
by only 0.4 kDa). However, when Mnn10p is shifted following the
addition of the tag, a band the size of Hoc1p remains. Interestingly in
the converse case, when the tag is attached to Hoc1p, only a very faint
band in the position of Mnn10p is observed. This is consistent with the
immunoprecipitation results described above, and with the notion that
the C terminus of Hoc1p may be close to the interface with Mnn10p.
Taken together, these results strongly suggest that there are two
different Mnn9p-containing complexes in the yeast cis Golgi. The first comprises Van1p and Mnn9p, and the second Anp1p, Mnn9p, Hoc1p, Mnn10p, and Mnn11p. Careful examination of the autoradiograms did not reveal any further unidentified bands in the Anp1p complex suggesting that these five proteins are the major constituents of the
complex. However it remains possible that there are further proteins
present at stoichiometry too low for detection by this approach. We
have previously shown that both these complexes have Analysis of M-Pol I and M-Pol II by Gel Filtration--
The above
analysis indicates that M-Pol II contains three more components than
M-Pol I. As such it would be expected to be a larger structure, and
this prediction was tested using gel filtration. The protease elution
method described above allows the complexes to be released from the
beads in an assembled state, and such released complexes were applied
to a gel filtration column. Fig. 4A shows that the two
complexes do indeed elute from such a column with very different
profiles. The Van1p complex (M-Pol I) elutes with a predicted size of
about 280 kDa, whereas the Anp1p complex (M-Pol II) is apparently
much larger with a size in excess of 1,000 kDa. The large size of M-Pol
II is unlikely to be a result of the precipitation and cleavage
protocol used to prepare it. When a whole cell lysate was applied to
the same column, Anp1p elutes in a similar position (Fig.
4B) and an epitope-tagged version of Mnn9p elutes in two
peaks corresponding to the positions of the isolated M-Pol I and M-Pol
II.
The individual predicted molecular masses of Van1p and Mnn9p total 107 kDa (plus the N-linked glycan on Van1p, which adds about 5 kDa to its apparent molecular weight on an SDS-polyacrylamide gel),
whereas those of the components of M-Pol II total 245 kDa. To maintain
the solubility of these membrane proteins the column was run in the
presence of digitonin, which forms micelles of 70 kDa in 0.1 M NaCl (33). Although this sort of gel filtration analysis
cannot be expected to give very accurate sizes, it seems likely that at
least some of the proteins are present in the M-pol complexes at a
greater than monomeric stoichiometry.
Deletion of Mnn10p and Mnn11p Results in Similar Defects in
N-linked Glycosylation--
It has been previously shown that M-Pol I
and M-Pol II have both
It has been observed previously that removal of the Mnn9p component of
M-Pol II results in the destabilization of Anp1p (12, 36). This
presumably reflects a requirement for association between the two
proteins for correct folding. In contrast, deletion of the other
members of the complex does not result in the complete loss of Anp1p
(Fig. 5B). However, the levels are reduced in some cases,
and normalization to a control ribosomal protein (Tcm1p) shows that in
the Deletion of Mnn10p or Mnn11p Alters the in Vitro
Mannosyltransferase Activity of M-Pol II--
Because the level of
Anp1p is reduced in the
The next step was to examine the activity of M-Pol II in the various
deletion strains. The amount of mannosyltransferase activity precipitated was greatly reduced in the In this paper we have examined the product of the
MNN10/BED1 gene. The gene was originally cloned
as corresponding to the bud emergence delay mutant bed1
(22). It was subsequently shown to correspond to the previously
identified mannan synthesis mutation mnn10 (26). Mnn10p is
distantly related to a protein Mnn11p, which we recently found to be a
component of one of the two protein complexes in the cis
Golgi that are involved in the synthesis of the backbone of yeast
mannan. Thus we examined its relationship to these two complexes, and
report here that it is a component of the Anp1p-containing complex that
we refer to as M-Pol II. It was initially reported that epitope-tagged
Bed1p was in the endoplasmic reticulum, based on overexpression of the
protein, but it was subsequently found to have a Golgi localization
when expressed as a single copy gene (22, 26). We have previously observed a similar phenomenon with another component of M-Pol II,
Anp1p, suggesting that both proteins need to be associated with other
components of the complex to fold correctly and exit the endoplasmic
reticulum (12, 37). Indeed deletion of MNN10 results in a
decrease in the levels of Anp1p.
The identification of Mnn10p as a component of M-Pol II brings the
number of proteins found in the complex to five. Co-immunoprecipitation suggests that all five are in the same complex, and indeed the apparent
size of the complex on gel filtration (~1,300 kDa) suggest that the
components are present in more than one copy per complex. This raises
the question of the exact function of the whole complex, and how this
is shared between the individual components. Previous work has shown
that deletion of either ANP1 or MNN1O results in defective mannan with a backbone reduced to 10-15 residues
(ANP1 being MNN8) (38,
39).3 We show here that
deletion of MNN11 results in an alteration in invertase
mobility similar to that seen with the deletion of these other two
genes. In contrast, it is known that deletion of MNN9, or of
VAN1, results in no mannan backbone at all beyond the single
-1,6-linked mannoses to which are attached many
branches consisting of
-1,2-linked and
-1,3-linked mannoses. The
initiation and subsequent elongation of the mannan backbone is
performed by two complexes of proteins in the cis Golgi. In
this study we show that the product of the MNN10/BED1 gene is a component of one of these
complexes, that which elongates the backbone. Analysis of interactions
between the proteins in this complex shows that Mnn10p, and four
previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are
indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis
in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p
are responsible for the majority of the
-1,6-polymerizing activity
of the complex.
INTRODUCTION
Top
Abstract
Introduction
References
-1,6-linked backbone of about 50 residues to which short branches of 3-4 residues in length are attached (1, 4, 5). The first 2 mannoses of these
branches are
-1,2-linked, and the final mannose is
-1,3-linked with some branches being additionally modified by the addition of a
phosphomannose residue. A similar type of mannan structure comprising a
long backbone with side branches is a feature of the cell wall of all
other yeast and fungi, although the precise linkages, and even sugar
residues, vary extensively even between quite closely related species
(6-10). This diversity may reflect a selective pressure to evade
hydrolytic enzymes or immune responses that recognize a particular
combination of residues and linkages.
-1,6-linked mannose residue of the mannan
backbone is attached to the core by the Och1p mannosyltransferase (11). This mannose is attached to all N-linked glycans, but then
only on a subset is it extended further to form the mannan backbone. On
the remaining glycans a single
-1,2-linked residue is added and then
an
-1,3-linked residue is added to this, and to other points on the
core structure (4). The extension of the mannan backbone is performed
by two recently identified complexes of proteins in the cis
Golgi. The first contains the proteins Mnn9p and Van1p, and the second
has been shown also to include Mnn9p as well as the proteins Anp1p,
Hoc1p, and Mnn11p (12). These two complexes possess
-1,6-transferase
activity in vitro, and mutations in their components can
result in either complete loss of the mannan backbone (mnn9
and van1) or mannan chains with a short backbone of 10-15
residues (anp1), suggesting that the two complexes act
sequentially to initiate and then extend the backbone. All of these
proteins are type II membrane proteins, with a single transmembrane
domain, a topology typical of Golgi glycosyltransferases (13, 14). The
branches on the mannan backbone are initiated and then extended by two
-1,2-mannosyltransferases encoded by MNN2 and
MNN5 (15). These two membrane proteins are not components of
the Van1p- or Anp1p-containing complexes, which make the backbone. Finally the
-1,3-linked mannose and the mannose phosphate are added
by Mnn1p and Mnn6p, respectively (16-18).
-1,6-polymerizing activity of
the complex.
EXPERIMENTAL PROCEDURES
ura3-52 his3
200 leu2-3,
112 trp-
901 lys2-801 suc2-
9)
and derivatives were used throughout (27). Genes were disrupted by
replacement of the open reading frame, or tagged at the exact C
terminus using homologous recombination with polymerase chain reaction
products containing Schizosaccharomyces pombe HIS5 or
Kluyveromyces lactis URA3 genes, and integrants checked with
polymerase chain reaction (28, 29). For protein A fusions cleavable by
the tobacco etch virus (TEV)1
protease, plasmid pZZ-HIS5 was used as described previously (15). For
hemagglutinin (HA) epitope tagging, polymerase chain reaction products
were amplified from plasmid p3xHA-HIS5 that encodes a linker of amino
acid sequence GAGAGA, followed by three copies of the sequence
YPYDVPDYA, with the first and third repeats followed by a G residue.
For Myc-tagging of open reading frames, a similar plasmid with nine
copies of the Myc tag was
used2 except for Myc-tagged invertase, which was expressed
in strains from a constitutive promoter using an integration plasmid
pTi Invmyc (26).
mnn11 and
mnn10 experiments) and analyzed
with a Molecular Dynamics PhosphorImager and ImageQuant software.
RESULTS
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Fig. 1.
Mnn1p associates with Anp1p but not with
Van1p. A, anti-HA-tag immunoblot of total protein from
yeast strain SEY6210 either with (M10-HA) or without
(wt) a triple HA tag at the end of the MNN10 open
reading frame. B, immunoblots of immunoprecipitates prepared
using the anti-HA monoclonal 12CA5 from the Mnn10-HA strain. Equivalent
amounts of immunoprecipitates (p), supernatants
(sn) and starting lysate (total) were probed with
antibodies to Anp1p and Van1p as indicated.
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Fig. 2.
Mnn10p, Mnn11p, and Hoc1p are all associated
with one another. A, immunoblots of proteins isolated
by immunoprecipitation from strains expressing Mnn10p and Mnn11p tagged
with either the Myc or HA epitopes as indicated. Proteins from
precipitations with mouse monoclonals against either Myc (M)
or HA (H), or the starting lysate (lys.) were
probed with a rabbit antiserum against HA. B, as shown in
A except that the strains expressed Myc-tagged Hoc1p and
either HA-tagged Mnn10p or HA-tagged Mnn11p, and precipitation was with
rabbit antisera against either Myc, HA, or Anp1p (A). The
blots were probed with monoclonal antibodies as indicated, and the
clipped form of Hoc1p is indicated by an asterisk.
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Fig. 3.
Precipitation of the Anp1p-containing complex
from labeled cells. A, autoradiogram of proteins
precipitated with IgG-Sepharose from lysates of yeast labeled with
[35S]methionine/cysteine. The yeast strains were either
SEY6210, or the same with a protein A tag attached to the C terminus of
either Anp1p or Van1p. The proteins were eluted from the beads using
TEV protease, which cleaves before the protein A tag, and then digested
with Endo H before polyacrylamide gel separation. B, as
shown in A except that the cleavable tag was attached to the
indicated protein, and the protease eluates run after treatment either
with or without Endo H. A 13-residue spacer remains after tag cleavage
resulting in the protein having a reduced mobility, and for the M-Pol
II components, this version of the protein is indicated by an
arrowhead. Mnn11p migrates as a slightly fainter and more
fuzzy band than the others, the former perhaps reflecting its having
fewer methionines than all but Hoc1p (12). Except for Van1p, none of
the other proteins is affected by Endo H digestion, consistent with
there being no predicted N-linked glycosylation sites in the
luminal domains of the five proteins (the site present, 8 residues into
the lumen from the TMD of Hoc1p, is predicted to be too close to the
membrane to be modified (46)).
-1,6-mannosyltransferase activity in vitro, attaching
multiple residues to make polymeric structures. To simplify further
discussion, we propose to refer to these two complexes as mannan
polymerases (M-Pol) I and II, respectively.
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Fig. 4.
M-Pol I and M-Pol II can be separated by gel
filtration. A, elution profile of immunoisolated M-Pol
I, or M-Pol II, from a Sephacryl S400 gel filtration column run as
described under "Experimental Procedures." The complexes were
detected by blotting with the indicated antiserum. Numbers indicate the
elution volume in milliliters after application of the sample.
B, elution profile of a digitonin lysate from a yeast strain
expressing HA-tagged Mnn9p, the fractions being analyzed by
immunoblotting with antisera to Anp1p or to HA to detect Mnn9p-HA.
C, elution profile of size standards on the gel filtration
column used in A and B, with the mobility of
M-Pol I (Van1p) and M-Pol II (Anp1p) also indicated.
-1,6- and
-1,2-mannosyltransferase
activity in vitro, raising the question of what contribution
the individual components of the complex make to this activity (12). To
address this issue, we initially examined the effect of deleting
individual subunits on mannan synthesis in vivo. It is known
that mutations in Mnn10p result in reduction in the length of mannan
chains, but the effect of loss of Mnn11p has not previously been
reported. The protein invertase is modified with mannan chains on 8-10
of its N-linked glycans, and Fig.
5A shows that the gel mobility
of invertase is increased in yeast in which either Anp1p, Mnn10p or
Mnn11p is absent. Consistent with this the
mnn11 strain
also shows increased sensitivity to hygromycin, a property of other
mannan defective mutants including anp1 and mnn10
(data not shown). This demonstrates that Mnn11p is required for the
synthesis of full-length mannan chains in vivo, and
justifies our inclusion of the gene in the MNN class as
defined by Ballou and co-workers (34, 35).
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Fig. 5.
Effect of the loss of Mnn10p or Mnn11p on the
glycosylation of invertase and on the levels of Anp1p.
A, immunoblot of total protein from yeast strain SEY6210
expressing a Myc-tagged version of invertase (wild type), or
the same strain in which the indicated genes were deleted, or SEY6210
without invertase ( ). B, immunoblots of total proteins
from strains, as shown in A, probed with antisera to Anp1p,
or with a monoclonal to the ribosomal protein L3 (Tcm1p) as a control
for protein recovery.
mnn10 and
mnn11 strains the level of
Anp1p is reduced by 85 and 75%, respectively, whereas deletion of
HOC1 has no effect. This suggests that Mnn10p and Mnn11p
contribute to the folding or stability of M-Pol II.
mnn10 and
mnn11
strains, it is possible that the defect in mannan synthesis seen is not
because the proteins are directly involved in mannan synthesis, but
rather because they are required for the stability or integrity of
other proteins in the complex, which are actually responsible for
mannan synthesis. To address the catalytic role of Mnn10p and Mnn11p,
the Anp1p-protein A fusion described above was used to isolate M-Pol II
for examination of its enzymatic activity in vitro. We
initially examined the activity of the M-Pol II from wild type cells,
and compared it with that of M-Pol I. Thus Anp1p and Van1p protein-A
fusions were precipitated from cells and incubated on the beads with
labeled GDP mannose and
-1,6-mannobiose as an acceptor. The products
of the transferase reactions were divided into aliquots and digested
with combinations of mannosidases. Finally, the charged fluorophore
ANTS was attached to the reducing ends of the products to allow
analysis by fluorophore-assisted carbohydrate electrophoresis (30). The
gels were autoradiographed and examined by fluorescence to examine the
reaction products. Fig. 6 shows that both
M-Pol I and M-Pol II produced a ladder of mannosylated products as
previously observed (12). Mannosidase digestion revealed that the
product of M-Pol II is almost completely digested to monomeric mannose
by
-1,6-mannosidase, suggesting that this is the major linkage made
by the complex. In contrast, for M-Pol I apparently only about half of
the products are
-1,6-linked.
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Fig. 6.
Mannosyltransferase activity of M-Pol I and
M-Pol II in wild type and mnn11 strains.
Fluorescence assisted carbohydrate electrophoresis gels of the products
of mannosyltransferase reactions using GDP-[14C]mannose
as a donor, and
-1,6-mannobiose as an acceptor. The reaction
products were digested with the indicated mannosidases, modified with
the charged fluorescent molecule ANTS, and then separated on
polyacrylamide gels. The resulting gels were exposed to a
PhosphorImager screen to identify the radioactive products (upper
panels) or visualized by fluorescence (lower panels).
The unmodified
-1,6-mannobiose substrate appears as a prominent band
in the fluorescent images and is completely digested by the
-1,6-mannosidase. A glucose oligomer standard ladder is visible in
the fluorescent images. For the mannosidase-treated samples the
percentage of radioactive sugar released as monomer was determined from
the phosphorimage and is indicated.
mnn10 and
mnn11 strains (~5-10% of wild type), but by using
twice as many cells and longer exposure times it was possible to
perform a digestion analysis. This revealed that in contrast to the
case for the intact complex, the reaction products had received just a
single mannose, and this was almost entirely resistant to
-1,6-mannosidase, even in conditions where the mannobiose substrate
was completely digested. Fig. 6 shows the results obtained with M-Pol
II from the
mnn11 strain, and a similar result was
obtained with the
mnn10 strain (data not shown). In
contrast, deletion of HOC1 did not alter the total activity
of M-Pol II, nor the size or digestion sensitivity of the reaction
product (data not shown). This result suggests that the reduction in
mannan synthesis seen in the
mnn10 and
mnn11 strains is not simply a consequence of a general
reduction in the level of the M-Pol II, but rather reflects the loss of the major
-1,6-mannosyltransferase activity of the complex.
DISCUSSION
-1,6-linked residue added by Och1p (38, 40). Thus it seems most
likely that the Mnn10p-containing M-Pol II elongates the mannan
backbone after it is initiated by the Mnn9p-Van1p-containing M-Pol I
(Fig. 7). In this model, M-Pol I would
perform the initial extension of the
-1,6-linked backbone beyond the
first
-1,6-linked residue, which is added to all N-linked
glycans by Och1p, consistent with the phenotypes of mnn9 and
van1 mutants. However, it should be noted that we found that
about half the mannose transferred in vitro by M-Pol I is in
a linkage that is resistant to
-1,6-mannosidase, but sensitive to
-1,2/3-mannosidase. This suggests that either the isolated M-Pol I
is associated with an additional protein with
-1,2/3-mannosyltransferase activity (although we have not found
association between M-Pol I and the
-1,2-mannosyltransferases Mnn2p,
Mnn5p and Mnt1p (12, 15)), or the complex itself can add
-1,2-mannose, as well as synthesizing the start of the
-1,6-linked backbone. One interesting explanation would be that the
-1,2-mannosyltransferase activity that is proposed to modify the
N-linked glycans that do not receive mannan, (
-1,2-MTII
in Fig. 7) could also reside in M-Pol I, a possibility that we are
currently investigating.
View larger version (25K):
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Fig. 7.
A model for the modification of
N-linked glycans in the Golgi of S. cerevisiae. All N-linked glycans have the
same Man8GlcNAc2 structure when they arrive at
the cis Golgi from the endoplasmic reticulum, and Och1p adds
a single -1,6-linked mannose to them all. However the next steps are
protein dependent. Many, but not all, of the proteins destined for
incorporation in or under the cell wall have a mannan structure
attached. The first step that commits them to this pathway is for M-Pol
I to attach a short
-1,6-linked mannose polymer to the mannose added
by Och1p. This is then extended further to a length of 40-60 mannoses
by M-Pol II. The extension of the backbone may then be terminated by a
capping
-1,2-linked mannose. The rest of the backbone is then
branched with
-1,2-linked mannoses by Mnn2p and Mnn5p, and the
chains terminated by an
-1,3-linked mannose added by Mnn1p, and
phosphomannoses added by Mnn6p. In contrast, the proteins of the
vacuole and the internal membranes, as well as some secreted proteins,
do not have mannan attached, but rather a single
-1,2-linked mannose
is added to the Och1p product by an as yet unidentified
-1,2-mannosyltransferase termed
-1,2-MTII (47). As discussed in
the text, this activity could lie within M-Pol I itself. Mnn1p attaches
-1,3-linked mannoses to this and to the core. What determines which
proteins receive which modification is at present unclear.
Although it seems likely that M-Pol II performs the extension of the
mannan backbone after it is initiated by M-Pol I, the question remains
of the functions of the individual components of M-Pol II. Mnn10p and
Mnn11p seem clear candidates to be -1,6-mannosyltransferases. First,
they contain a "DXD" motif that is conserved in many of their homologues that we have recently shown is a common feature of
many families of nucleoside-diphosphate-sugar using
glycosyltransferases (17). Second, when assayed in vitro the
isolated M-Pol II catalyzes primarily the formation of
-1,6-linkages
with only a small percentage of the product being susceptible to
-1,2/3-mannosidase, but when either Mnn10p or Mnn11p is removed
there is relatively much less
-1,6-transferase activity in the
complex. It may be that having two such transferases, possibly present
in multiple copies, in a large complex facilitates the rapid synthesis
of a long
-1,6-linked backbone. One objection to this model might be
that Mnn10p is quite closely related to an
-1,2-galactosyltransferase from S. pombe (GM12) (41). The
conserved portion of Mnn10p (the C-terminal 280 residues) shows 27%
identity to GM12 and only 21% identity to Mnn11p, when aligned with
PSI-BLAST (42). However it is very unlikely that Mnn10p is a
galactosyltransferase. Galactose is not normally found in S. cerevisiae glycoproteins, and indeed when the S. pombe
GM12 is expressed in the cells, galactose is now incorporated into the
mannan (43). The relatedness of the two genes may reflect evolutionary
pressure to vary mannan structure. Branched polysaccharides containing
either mannose alone, or mannose and galactose (galactomannans) are a
common feature of the cell walls of yeast and fungi (44, 45). It is
possible that the pressure for variation of cell wall structure has
caused the enzymes to shift between using galactose and mannose, and to
adding either the backbone or the branches with different linkages. The
presence of homologues of Mnn10p in plants and Caenorhabditis
elegans raises the possibility that related complexes of
glycosyltransferases may be involved in making large oligosaccharide
structures in other organisms (12, 26).
If Mnn10p and Mnn11p are -1,6-mannosyltransferases, then what can be
said of the function of Anp1p, Mnn9p, and Hoc1p? All have conserved
DXD motifs, and Hoc1p is homologous to the Och1p
-1,6-mannosyltransferase. Although they may all contribute to
-1,6-mannosyltransferase activity, we also found that M-Pol II can
form linkages resistant to
-1,6-mannosidase but sensitive to an
-1,2/3 mannosidase. This is unlikely to represent the initiation of
the
-1,2-linked branches as we have recently identified the product
of the MNN2 gene as being responsible for this, and it is
not a component of M-Pol II (15). However, in mnn2 strains, a single
-1,2-linked mannose is still found at the very end of an
otherwise unbranched chain, and it has been suggested that it might
serve a capping function (39). This capping mannose could be added by
one of these remaining components and it may serve to regulate mannan
size, or prevent re-elongation of the products of M-Pol II. It has also
been argued that Hoc1p has a regulatory function as removal of the gene
has little effect on mannan size, but overexpression can suppress
defects in protein kinase C (20). It has long been suggested that the
size of mannans is regulated in a cell cycle and growth
state-dependent fashion. Now that a clearer picture of the
composition of M-Pol I and M-Pol II is emerging it will hopefully be
possible to understand the precise structure, functioning, and
regulation of these complexes, which may reveal principles relevant to
the synthesis of large oligosaccharides in other eukaryotes.
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ACKNOWLEDGEMENTS |
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We thank Neta Dean and Jon Warner for providing reagents and advice, Clint Ballou for making all this possible, and Hugh Pelham, Tim Levine, and James Whyte for critical reading of the manuscript and helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Tel.: 00-44-1223-402236; Fax: 00-44-1223-412142; E-mail: sean{at}mrc-lmb.cam.ac.uk.
2 I. Adams and J. V. Kilmartin, unpublished material.
3 Neta Dean, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: TEV, tobacco etch virus; HA, hemagglutinin; Endo H, endoglycosidase H; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; M-Pol, mannan polymerase.
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REFERENCES |
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