From the Department of Biomedical Sciences, State
University of New York at Albany, Albany, New York 12201, the
§ Wadsworth Center, New York State Department of
Health, Albany, New York 12201-0509, and the ¶ Department of
Biochemistry and Cell Biology, Institute for Cell and Developmental
Biology, State University of New York at Stonybrook, Stonybrook, New
York 11794-5215
Received for publication, December 4, 2000, and in revised form, February 13, 2001
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ABSTRACT |
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The initial steps in N-linked
glycosylation involve the synthesis of a lipid-linked core
oligosaccharide followed by the transfer of the core glycan to nascent
polypeptides in the endoplasmic reticulum (ER). Here, we describe
alg11, a new yeast glycosylation mutant that is defective
in the last step of the synthesis of the
Man5GlcNAc2-PP-dolichol core oligosaccharide on
the cytosolic face of the ER. A deletion of the ALG11 gene
leads to poor growth and temperature-sensitive lethality. In an
alg11 lesion, both Man3GlcNAc2-PP-dolichol and
Man4GlcNAc2-PP-dolichol are translocated into
the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family
of oligosaccharide structures lacking one or both of the lower arm
Asparagine-linked (N-linked) glycosylation is an
essential and highly conserved modification that is required for the
function of glycoproteins in eukaryotic cells. Although
N-linked oligosaccharides represent a diverse group of
structures, all share a common biosynthetic pathway that begins with
the synthesis of a core oligosaccharide consisting of
Glc3Man9GlcNAc2 (for reviews
see Refs. 1 and 2). The assembly of the core oligosaccharide precursor
occurs in two compartments. On the cytosolic face of the
ER,1
Man5GlcNAc2 is assembled stepwise from the
nucleotide sugars UDP-GlcNAc and GDP-Man on to a lipid carrier,
dolichylphosphate (3, 4). A "flipping" of the lipid-linked
precursor translocates the oligosaccharide-lipid (OSL) into the lumen
of the ER. Here, the next seven sugars (four Man and three Glc
residues) are donated from Man-P-Dol and Glc-P-Dol, respectively, to
form Glc3Man9GlcNAc2. As a nascent
polypeptide passes into the lumen of the ER, the preassembled
oligosaccharide is transferred as a unit to selected asparagine
residues in a reaction catalyzed by the enzyme
oligosaccharyltransferase (OST). After its transfer, the
oligosaccharide undergoes further processing in the ER by glucosidases
I and II, and in Saccharomyces by an Mutants from the budding yeast, Saccharomyces
cerevisiae, have been isolated that are defective in various steps
involved in the synthesis of the
Glc3Man9GlcNAc2 core.
Characterization of the alg
(asparagine-linked glycosylation)
mutants demonstrates that the early steps of core assembly are
essential, whereas the later steps are not (7). Yeast with conditional
mutations that interfere with the synthesis of the nucleotide sugar
donors or the early biosynthetic steps that occur on the cytoplasmic
face of the ER accumulate highly truncated core oligosaccharides and define essential genes, including DPM1 (8), SEC59
(9), ALG1 (10, 11), ALG2 (12), and
ALG4 (13). In contrast, mutations in genes that accumulate
lipid-linked core oligosaccharides in which five or more mannose
residues have been attached or that are defective in the later
processing reactions, including ALG3 (7, 14),
ALG5 (7, 15), ALG6 (7, 16), ALG8 (17), ALG9 (18), and ALG10 (19), show little or no
growth phenotype. Truncated oligosaccharides are transferred by OST to
proteins in vivo (11, 20) and in vitro (21-23),
though the full-length, glucosylated
Glc3Man9GlcNAc2 is the preferred
substrate (21, 24). Although the transfer of truncated oligosaccharide
structures is tolerated, mutations that block OST activity are
lethal (25), demonstrating that N-linked glycosylation is an
essential function.
Although considerable progress has been made in the isolation of genes
encoding proteins that mediate some of these stepwise reactions, the
regulation of glycosylation in the ER is still not fully understood.
This report describes the characterization of ALG11, a yeast
gene that is required for normal glycosylation and is essential for
growth at high temperatures. The alg11 mutant was originally
recovered based on its resistance to sodium vanadate, a drug that
enriches for glycosylation mutants (26, 27). Oligosaccharide structural studies define the alg11 defect to be the final
step in Man5GlcNAc2-PP-Dol synthesis on the
cytosolic face of the ER, specifically the addition of the terminal
Yeast Strains, Media, and Genetic Methods--
Yeast strains
were grown in either YPAD (1% yeast extract, 2% peptone, 2%
dextrose, 50 mg/liter of adenine sulfate) or synthetic medium that
contained 0.67% yeast nitrogen base and 2% glucose, supplemented with
the appropriate auxotrophic requirements (28). YPAD liquid medium was
supplemented with 0.5 M KCl for the growth of
alg11 mutant strains, which are osmotically sensitive.
Hygromycin B (Roche Molecular Biochemicals) was added to YPAD agar
after autoclaving to a final concentration of 30 µg/ml.
All yeast strains used in this study are listed in Table
I. NDY13.4, which carries the
alg11-1 allele, was isolated as a spontaneous
vanadate-resistant mutant on YPAD plates containing 7.5 mM
sodium orthovanadate (Fisher) (27). NDY41, containing a deletion of the
ALG3 gene, was constructed by replacing the ALG3
gene in SEY6210, using the linearized disruption plasmid pYBL0720::HIS3 (kindly provided by M. Aebi and
S. te Heesen) as described (14). Yeast strains were transformed using
the lithium acetate procedure (29).
Isolation of the ALG11 Gene--
Strain NDY13.4 was transformed
with a yeast genomic CEN-based library in YCp50, carrying
the URA3 selectable marker. Prototrophic transformants were
selected on media lacking uracil. These transformants were replica
plated on media containing 30 µg/ml of hygromycin B. Plasmid
DNA from hygromycin B-resistant colonies was isolated, amplified in
Escherichia coli, and retransformed into the
alg11-1 mutant to confirm the hygromycin B resistance.
A 5.2-kb SacI/ClaI fragment containing the
ALG11 complementing activity was subcloned into pRS316 (30)
to generate a URA3, CEN-based plasmid, p6DB. This
fragment contains the entire ALG11 gene and regulatory
sequences, as well as an upstream ORF. The 5' and 3' ends of this
fragment were sequenced by the dideoxy method (31) and were used to
search the S. cerevisiae genome data base to obtain the
entire nucleotide sequence corresponding to this
SacI/ClaI fragment. This fragment contains two
ORFs. Each of these ORFs was subcloned and tested for the ability to
rescue the hygromycin B sensitivity of alg11 to identify the
complementing activity. Searches of the data bases were made using the
BLAST algorithm (32), and sequence alignments were made with the
MegAlign program (DNASTAR) using the Clustal algorithm.
Plasmid Constructions--
All DNA manipulations were carried
out according to standard protocols (33). A 2.2-kb NheI/ClaI
fragment was isolated from the plasmid p6DB. This fragment, which
contains only the ALG11 ORF and 560 base pairs of upstream
flanking sequence, was subcloned into pBR322 to produce pBRNhe/Cla. A
2.2-kb BamHI/ClaI fragment containing the
ALG11 ORF and upstream regulatory sequences was subcloned
into pRS316 (30) to produce pALG11-316 and into Bluescript SK
The deletion plasmid, palg11
The integrative plasmid, pALG11-306, was constructed by ligating the
2.2-kb BamHI/ClaI fragment from pALG11-316 into
pRS306 (30). The plasmid was linearized at a unique EcoRI
site in the ALG11 gene for integration at the
ALG11 locus. The ALG11 overexpression plasmid, pYEp24-ALG11, was constructed by ligating the 2.2-kb BamHI/SalI fragment from pAlg11-316 into the
URA3, 2µ plasmid, YEp24.
An epitope-tagged version of ALG11, pALG11-HA, was
constructed by using polymerase chain reaction to introduce a
HindIII site at the 5' end and an NsiI site at
the 3' end of the gene that replaces the stop codon of the
ALG11 ORF with a cysteine codon. The 1.6-kb
HindIII/NsiI fragment containing the
ALG11 ORF was cloned into pSK Western Immunoblotting and Immunofluorescence--
Overnight
yeast cultures were diluted to 107 cells/ml and grown for 3 to 4 h prior to protein extraction. Whole cell protein extracts
were prepared as described (35). For removal of N-linked oligosaccharides, extracts were resuspended in 0.1% SDS, 1%
2-mercaptoethanol, 50 mM sodium acetate and boiled for 3 min. After the addition of phenylmethylsulfonyl fluoride to 0.5 mM, 1 milliunit of endoglycosidase H (endo H; New England
Biolabs) was added, and samples were incubated for 60 min at 37 °C.
Proteins were separated by 8% SDS-PAGE and immunoblotted as described
(35). Anti-CPY antibodies were used at a 1:3000 dilution. Culture
supernatants containing the monoclonal anti-HA antibody, 12CA5, were
used at a 1:10 dilution. Anti-chitinase antibodies were used at a
1:1000 dilution. Secondary anti-rabbit or anti-mouse antibodies,
conjugated to horseradish peroxidase (Amersham Pharmacia Biotech), were
used at a 1:3000 dilution and detected by chemiluminescence (ECL;
Amersham Pharmacia Biotech) followed by autoradiography. Indirect
immunofluorescence of yeast cells expressing Alg11-HAp and data
analyses were as described (36).
Isolation of Lipid-linked and Whole Cell Pellet
Oligosaccharides--
Mannosylated and glucosylated in vivo
alg11 Chromatography--
Oligosaccharides, released from
alg11 Isolation of Oligosaccharides from alg11 Mass Spectrometry--
MALDI-TOF mass spectrometry was performed
on a Bruker reflex instrument. Samples of 25 to 50 pmol were
co-crystallized with 2,5-dihydrobenzoic acid as the matrix. Data from
10 to 50 3-ns pulses of the 337-nm laser were averaged for each sample.
Analyses were performed in linear and reflective mode.
Methylation Linkage Analysis--
Samples were analyzed by
methylation as described (41) using the NaOH/Me2SO
method. Briefly, the free hydroxyls of the oligosaccharides were
deprotonated with NaOH/Me2SO and then CH3I was
added to replace the free hydroxyls with methoxy groups. The
methoxylated oligosaccharide was hydrolyzed in strong acid, evaporated
under low pressure, and applied to Whatman silica gel 60A TLC plates.
The plates were developed twice with MeCN-CHCl3-MeOH, 3:9:1
(v/v/v), thoroughly dried between each ascent, and rapidly dipped into
a solution containing 3 g of
N-(naphthyl)ethylenediamine and 50 ml of concentrated H2SO4 in 1 liter of CH3OH. The
plates were dried and placed in an oven for 10 min at 120 °C. All
saccharide standards were purchased from Sigma.
1H NMR Spectroscopy--
Oligosaccharides (0.15-1.0
pmol) were exchanged three times by rotary evaporation from 99.8%
D2O and twice by lyophilization from 99.96%
D2O. Lyophilized samples were dried over
P2O5 in vacuo for a day or more and
then reconstituted in 0.5 ml of 99.996% D2O (containing
1.35 mM acetone as an internal chemical shift reference) to
a final concentration of 0.3-2 mM. Acetone protons were
set at 2.225 and 2.217 ppm for spectra taken at 300 and 318 K,
respectively. Samples were quickly transferred to 5-mm tubes (catalog
number 535pp; Wilmad Co., previously washed and exchanged with
99.8% D2O), flame-sealed, and examined at 300 and 318 K by 1D and 2D DQF-COSY phase-sensitive 1H NMR spectroscopy at
500 MHz as described previously (42-44). Spectral width in the
11.7-tesla field was 1502 Hz for all experiments. For acquisition of 1D
data, 1024 scans were collected over 4096 data points. The limit of
resolution was 0.0045 ppm based on the ratio of the width of the widest
peak at half-height (2.26 Hz) over the number of Hz per ppm (500.13 Hz/ppm). For homonuclear 2D DQF-COSY, 1.5 s of low power
presaturation on residual HDO at 4.79 ppm was applied in the 300 K
experiments. Data collection for the 2D experiments was 4096 data
points in t2 and 512 complex data points in the
indirect t1 dimension. The 2D relayed ROESY experiments were conducted at 318 K as described by Cipollo
et al. (45).
The alg11 Mutation Affects the Synthesis of N-Linked
Glycoproteins--
Resistance to sodium vanadate has been described
previously as a way to isolate yeast glycosylation mutants (26, 35), and using this selection, a new mutant, alg11, was
identified that affects an early step in glycosylation. The
glycosylation state of the vacuolar proteinase, CPY, which undergoes a
series of modifications as it transits the secretory pathway, is a
useful probe to assess normal versus aberrant glycosylation
(17-19). The 67-kDa ER form of CPY contains four core
N-linked glycans, the 69-kDa Golgi form is further modified
by the addition of sugars, and in the vacuole CPY is proteolytically
processed to the mature 61-kDa form. Mutations that affect the assembly
or transfer of oligosaccharides to CPY result in underglycosylation,
which is readily observed as an increased mobility in which forms with 4, 3, 2, 1, or 0 N-glycans form a ladder of bands on
SDS-PAGE (46). Lysates were prepared from alg11-1 and
wild-type cells, separated by SDS-PAGE, and assayed by immunoblotting
using anti-CPY antiserum.
The alg11-1 mutant accumulated CPY as a series of
intermediates that migrated with an increased mobility compared with
that of the mature wild-type form (Fig.
1A). The mobility of CPY in alg11-1 was compared with that in the ost4-2
mutant. OST4 encodes a component OST (47) and when
defective, reduces the transfer of N-linked oligosaccharides
to CPY in the ER (47). Both alg11-1 and ost4-2
reduce the N-glycosylation of CPY, but the defect was more
restrictive in alg11-1, as a higher proportion of forms
with only one or two glycans was apparent (Fig. 1A, compare
lanes 2 and 3). ALG11 and
alg11-1 CPY treated with a low concentration of endo H
resulted in identical deglycosylated products (Fig. 1B).
This result confirms that the increased mobility of CPY in alg11-1 extracts is because of underglycosylation and not
proteolysis and that the majority of the oligosaccharides of CPY have
the middle arm
In addition to N-linked glycosylation, yeast also
initiate O-linked glycosylation in the ER (reviewed in
Ref. 49). To assay whether the alg11 mutation affected
O-linked carbohydrates, the glycosylation state of chitinase
was examined. Because chitinase contains carbohydrates that are
exclusively O-linked to Ser or Thr residues, an effect on
O-linked glycosylation can be detected by an increase in its
mobility by SDS-PAGE (50). A comparison of wild-type and
alg11-1 chitinase, assayed by immunoblotting cell extracts
separated by SDS-PAGE with anti-chitinase antiserum, is shown in Fig.
1C, lanes 1 and 2. As a control, these
chitinases were compared with that from vrg4-2 cells, a
mutant affected in the lumenal transport of GDP-mannose into the Golgi
and therefore defective in O-glycan elongation (35, 36).
Although an increase in chitinase mobility was detected in the
vrg4-2 mutant (Fig. 1C, lane 3), no
such difference was observed between ALG11 and alg11-1 chitinase (Fig. 1C, compare lanes
1 and 2). These results suggest that alg11
does not affect O-linked glycosylation.
In addition to O-linked sugar addition, no effect of the
alg11 mutation was observed on the addition of
glycosylphosphatidylinositol anchors, the steady-state level of
Man-P-Dol, or in vitro OST activity (data not shown). Thus,
these results suggest that ALG11 is specific for a step in
the N-linked glycosylation pathway.
Isolation of the ALG11 Gene--
The alg11 mutant phenotype
of severe under N-glycosylation of CPY, but with endo
H-sensitive glycans, suggested the ALG11 locus might encode
one of the few OSL biosynthetic steps remaining to be identified. Like
many other yeast glycosylation mutants, alg11 growth is
completely inhibited on medium containing hygromycin B, a condition
that does not restrict the growth of wild-type cells (see Ref. 27 and
Fig. 2). The glycosylation defect and hygromycin B sensitivity co-segregated through two sequential out
crosses, and the mutation behaved as a single recessive allele (data
not shown). Therefore, we cloned the wild-type ALG11 gene by
complementation of the hygromycin B sensitivity (see "Experimental Procedures"). The alg11 complementing activity was
localized to YNL048w, an ORF of previously unknown function, which we
designate ALG11 (for
asparagine-linked glycosylation). A
plasmid containing only this ORF expressed in the alg11-1
mutant rescued the growth defect (data not shown), the glycosylation
defect as assayed by CPY immunoblots (Fig. 2A), and the
hygromycin B sensitivity (Fig. 2B).
To confirm that the cloned fragment contained the ALG11
locus, rather than an extragenic suppressor, a fragment containing ALG11 was cloned into an integrative plasmid that carries
URA3 as a selectable marker. The plasmid was linearized at a
unique site within the putative ALG11 gene to allow
homologous recombination at the ALG11 locus in an
ALG11 ura3-52 strain. Ura+ transformants were
crossed to an alg11-1 ura3-52 strain to produce diploids
that were sporulated and dissected. A 2:2 segregation pattern was
observed for hygromycin-sensitive: hygromycin-resistant colonies, and all of the hygromycin-resistant spores were
Ura+ in the 25 tetrads analyzed (data not shown). These
results confirm that the cloned DNA is very tightly linked to the
alg11 mutation and suggest that this DNA fragment does
indeed contain the ALG11 gene.
Alg11p Is Highly Conserved among Eukaryotes, Is Related to Alg2p,
and Contains a Conserved Motif Found in Many Sugar Binding
Enzymes--
The nucleotide and predicted protein sequence of
ALG11 is deposited in GenBankTM
(accession number U12141). The ALG11 gene encodes a
predicted protein of 548 amino acids with a molecular mass of 63.1 kDa. Hydrophobicity analysis predicts that the protein contains at least
three membrane-spanning domains. A search of the data bases (32)
identified five other proteins that displayed significant homology
along their lengths to Alg11p, whose alignment is shown in Fig.
3A. These included homologues
from Schizosaccharomyces pombe (51), Caenorhabditis
elegans, Drosophila melanogaster, Leishmania
major, and Arabodopsis thaliana.
Other proteins displaying a more limited but still significant homology
to Alg11p were also identified, corresponding to Alg2p from S. cerevisiae and C. elegans (Fig. 3B).
ALG2 encodes a protein that regulates or catalyzes an early,
cytoplasmically oriented step in the biosynthesis of the core
oligosaccharide, the addition of
All of the Alg11- and Alg2-related proteins share a
sequence, beginning at position 404 of the S. cerevisiae
Alg11p, that is particularly well conserved. When this sequence was
used to search the data base, over 60 related proteins were identified, a subset of which is shown in Fig. 3C. These proteins, which
include members from bacteria to man, perform functions relating to
sugar metabolism. Included in this group of proteins are various
glycosyltransferases, sucrose synthases, proteins that function in
bacterial lipopolysaccharide biosynthesis, and proteins that function
in glycosylphosphatidylinositol anchor biosynthesis. This high degree
of conservation implies that this sequence may represent a domain
required for some aspect of carbohydrate binding or metabolism. Though
speculative, these proteins all utilize nucleotide sugar substrates,
suggesting that this motif may correspond to a nucleotide
sugar-recognition site.
Disruption of the ALG11 Locus Results in Underglycosylation of
Proteins and Temperature-sensitive Lethality--
The
alg11-1 mutation leads to a severely impaired growth
phenotype. Other glycosylation mutants that are blocked in the lumenal steps of core biosynthesis display no obvious growth phenotype. For
instance, deletion of ALG3 and ALG9, which block
addition of the sixth and seventh Man to
Man5GlcNAc2-PP-Dol, respectively, and
ALG6, which blocks addition of the first Glc to
Man9GlcNAc2-PP-Dol, have no apparent effect on
growth rate (14, 16, 18). The impaired alg11-1 growth
phenotype in conjunction with the sequence similarity between Alg11p
and Alg2p suggested that Alg11p might be a mannosyltransferase involved
with the cytosolic synthesis of
Man5GlcNAc2-PP-Dol.
To further investigate this mutant, the phenotype of an
alg11 null allele was determined. An 832-base pair
EcoRV fragment within the ALG11 gene was replaced
with a fragment encoding the URA3 gene. A standard one-step
gene disruption (52) was performed to replace the chromosomal copy of
ALG11 with the null allele in a diploid, as described under
"Experimental Procedures." The disruption of one allele was
confirmed by polymerase chain reaction analysis of genomic DNA (data
not shown). Heterozygous diploids were sporulated, and 15 dissected tetrads were analyzed for cell viability at
25 °C (Fig. 4A). Colonies
from spores carrying the null allele that were Ura+ could
not be detected until 5 days after the wild-type colonies arose,
indicating that the slow growth phenotype of the null allele was much
more severe than the alg11-1 allele and that the severe growth defect is directly because of the alg11 mutation.
Furthermore, no growth was observed when
alg11 ALG3 Is Not Epistatic to ALG11--
As a further genetic test of
whether ALG11 might function in the cytosolic assembly of
Man5GlcNAc2-PP-Dol, the phenotype of an
alg3 Alg11p Is Localized in the ER--
To examine the physical
properties of the Alg11 protein, an epitope-tagged ALG11
allele was constructed that encodes three tandem copies of the HA
epitope at the C terminus. Expression of the Alg11-HAp fusion protein
in an alg11
The intracellular location of Alg11-HAp was analyzed by indirect
immunofluorescence in wild-type cells. A perinuclear pattern of
fluorescence (Fig. 5B) characteristic of the ER in yeast
co-localized with DAPI-stained nuclei (not shown). In conjunction with
fractionation experiments that demonstrated Alg11p is insoluble and
associated with a membrane fraction (data not shown), these results
suggest that Alg11p is a membrane protein that resides in the ER.
N-Linked Oligosaccharide Structures in alg11
Digestion of Hex6GlcNAc2 (Fig. 6A,
peak b) with
The [3H]Man-labeled Hex7GlcNAc2
represented ~80% of the glycans released from pulse-labeled
alg11
The Hex11GlcNAc2 pool (Fig. 6A,
peak d) lost the equivalent of two hexoses on
3H-Glc- and 3H-Man-labeled N-Linked Glycan
Structures on alg11
The above experiments with labeled Glc and Man indicate that two series
of truncated N-glycans arise in the alg11 Assigning alg11
To determine how N-glycans were processed in
alg11 alg11
If this occurred, we would expect to find essentially all of
residue 5 substituted by
MALDI-TOF MS provided a distribution of Hex30-49GlcNAc
with an average size of Hex39GlcNAc for the
alg11 In this report we describe the characterization of
alg11, a yeast mutant that is deficient in the assembly of
lipid-linked oligosaccharides, which was isolated as a sodium
vanadate-resistant, hygromycin B-sensitive glycosylation mutant (27).
Sodium vanadate is a drug that enriches for yeast glycosylation mutants
(26, 27), although the molecular mechanism for this enrichment is not
understood. The severely impaired growth phenotype associated with
alg11 The absence of the terminal The accumulation of some Man3GlcNAc2-PP-Dol on
steady-state labeling of alg111,2-linked Man residues. The former are elongated to mannan, whereas
the latter are poor substrates for outerchain initiation by Ochlp
(Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and
Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate
largely as truncated biosynthetic end products. The ALG11
gene is predicted to encode a 63.1-kDa membrane protein that by
indirect immunofluorescence resides in the ER. The Alg11 protein
is highly conserved, with homologs in fission yeast, worms, flies, and
plants. In addition to these Alg11-related proteins, Alg11p is also
similar to Alg2p, a protein that regulates the addition of the third
mannose to the core oligosaccharide. All of these Alg11-related
proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism,
implicating this sequence as a potential sugar nucleotide binding motif.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,2-mannosidase,
Mns1p, that removes a single specific residue (5) as part of the
editing mechanism that promotes the exit of correctly folded
glycoproteins from the ER (6).
1,2-linked Man to Man4GlcNAc2-PP-Dol. As a
consequence of the elongation of
Man3,4GlcNAc2-PP-Dol in the ER lumen by
subsequent Man-P-Dol-dependent transferases,
Man7GlcNAc2-PP-Dol accumulates. Though the size
of this oligosaccharide implies that it is the addition of the eighth
mannose that is blocked in alg11 strains, other
alg mutants that accumulate more highly truncated oligosaccharides display no growth phenotype. Thus, the
temperature-sensitive lethality of alg11, as well as genetic
epistasis analyses reported here, are fully consistent with a role for
ALG11 during the earliest steps of N-linked glycosylation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in the present study
(Stratagene) to produce pALG11-SK
.
::URA3, was constructed by
replacing an internal 842-base pair EcoRV fragment within
the ALG11 gene with a 2-kb SmaI/SalI
fragment containing the URA3 gene. Replacement of the
EcoRV fragment results in removal of approximately half of
the ORF, after the first 95 amino acids.
P/X HA3 (34),
resulting in the in-frame fusion of ALG11 to sequences encoding three copies of the HA epitope at the C terminus, in the
PstI/XbaI site of pBlueScript SK
(Stratagene). For its expression in yeast, the HA-tagged
ALG11 gene, on a HindIII/SacI
fragment, was cloned into the HindIII/SacI site
of pRS425-TPI. pRS425-TPI was constructed by ligating a 1.2-kb SalI/HindIII fragment that contains the
TPI promoter into the SalI/HindIII site of pRS425 (30). This places
ALG11 under the control of the strong, constitutive
TPI promoter in a 2µ, LEU2 yeast shuttle vector.
cells were grown overnight in YPD + 0.5 M KCl
to a density of 4 × 107 cells/ml and collected by
centrifugation for 5 min at 3000 rpm and room temperature. The yeast
were washed twice in YP + 0.1% glucose medium by centrifugation, and
cells (1 × 109) were resuspended in 200 µl of YP + 0.1% glucose containing 250 µCi of either [2-3H]Glc
(15 Ci/mmol) or [2-3H]Man (20 Ci/mmol) purchased from
American Radiolabeled Chemicals, St. Louis, MO. The labeling and
preparation of OSLs were performed as described previously (11) with
the following modifications. Pulse labeling was for 2 min, and
steady-state labeling was for 15 min. Labeling was terminated by
addition of 4 ml of CHCl3/CH3OH (3:2). To
isolate N-linked oligosaccharides, whole cell pellets were
solubilized in 1% SDS in 50 mM sodium phosphate buffer (pH 8.5) with heating, followed by pH adjustment to 5.5 with 1.0 M phosphoric acid, and endo H was then added to 50 milliunits/ml. The reactions were incubated at 37 °C for 16 h,
and endo H activity was verified by hydrolysis of
Man6GlcNAc4-Asn-dansyl, followed by paper
chromatography of the released GlcNAc-Asn-dansyl moiety. The released
glycans were solvent-precipitated in 80% acetone as described
previously (20).
OSL or glycoproteins (see below), were sized on a
calibrated Bio-Gel P-4 column (1.6 × 96-cm) eluted at room
temperature with 0.1 N CH3COOH/1%
n-butanol (20). Fractions of 0.74 ml were collected and
scanned for radioactivity by scintillation counting or for neutral
hexose by a modification (5) of the phenol sulfuric acid assay
(37).
Cells for Structural
Studies--
Two separate 200-g batches of logarithmic phase cells
were suspended in two volumes of 50 mM sodium citrate, pH
5.5, broken with 0.5-mm glass beads in a Bead Beater (Bio-Spec
Industries, Bartlesville, OK), and a crude cell extract was prepared as
described (20). Solid ammonium sulfate was added to the extract to 50% saturation, stirred for 30 min at 4 °C after the salt was in
solution, and centrifuged at 30,000 × g for 15 min at
4 °C. The supernatant fraction (400-ml) was dialyzed over 72 h
at 4 °C against three 6-liter changes of 5 mM sodium
citrate, pH 5.5, and concentrated with an Amicon spiral cartridge
concentrator to 80 ml. After lyophilization, the sample was taken up in
15 ml of dH2O, and protein was determined by the Bearden
assay (38). Glycoproteins were denatured with a 1.2-fold weight excess
of SDS (39), and oligosaccharides were hydrolyzed with endo H (40) at
50 milliunits/ml overnight at 30 °C. Protein and glycans were
precipitated at
20 °C by the addition of four volumes of cold
acetone, and the oligosaccharides were recovered from the pellet by
extraction with 60% aqueous methanol as described (39). Methanol was
removed by rotary evaporation under reduced pressure, and the
oligosaccharides were taken up in 1.5 ml of 0.1 N
CH3COOH and initially chromatographed on a preparative
Bio-Gel P-4 column (2.6 × 67-cm) with a typical profile shown in
Fig. 7. Fractions (1.35-ml) corresponding to Hex5,7,9GlcNAc (odds) and Hex6,8GlcNAc (evens) from the two preparative
columns were pooled separately, and each was rechromatographed into
isolated glycan sizes on the calibrated 1.6 × 96-cm Bio-Gel P-4
column described above (not shown). The central 85% of the
phenol-sulfuric acid assay color for each peak was collected as pools
I-V corresponding to Hex5-9GlcNAc, respectively, and
the glycan eluting in the column void- volume was collected as
pool VI representing "mannan."
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-linked Man added to the
Man5GlcNAc2-PP-Dol core OSL by Alg3p in the ER
lumen, a requirement for endo H sensitivity (48).
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Fig. 1.
The alg11 mutation affects
the synthesis of N-linked glycoproteins.
A, Western immunoblot analysis of carboxypeptidase Y in
alg11 mutant and wild-type cells. Proteins were extracted
from the isogenic parental cells ALG11 (MCY1094) (lane
1), alg11-1 (NDY13.4) (lane 2), or
ost4-2 (NDY17.4) (lane 3), separated by 8%
SDS-PAGE, and subjected to immunoblot analysis using anti-CPY
antibodies as described under "Experimental Procedures." The
arrow denotes the mobility of mature CPY. B,
protein extracts were prepared from ALG11 (MCY1094)
(lanes 1 and 2) or alg11-1 (NDY13.4)
(lanes 3 and 4) yeast strains and incubated in
the presence (lanes 2 and 4) or absence
(lanes 1 and 3) of endo H as described under
"Experimental Procedures." Proteins were separated by SDS-PAGE and
subjected to immunoblot analysis with anti-CPY antibodies as in
Panel A. The arrows denote the position of mature
CPY (mCPY) or deglycosylated CPY (dgCPY).
C, immunoblot analysis of chitinase in alg11
mutant and wild-type cells. Proteins were extracted from the
supernatants of ALG11 (MCY1094) (lane 1),
alg11-1 (NDY13.4) (lane 2), or
vrg4-2 (NDY5) (lane 3) cultures, separated by
8% SDS-PAGE, and subjected to immunoblot analysis using anti-chitinase
antisera as described under "Experimental Procedures." The
arrow denotes the mobility of chitinase.
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Fig. 2.
The cloned ALG11 gene
rescues both the glycosylation defect and the hygromycin B sensitivity
of alg11-1. A, immunoblot analysis of
CPY, using extracts prepared from wild-type (SEY6210),
alg11-1 (NDY13.4), or alg11-1 cells harboring a
plasmid bearing the wild-type ALG11 gene (NDY13.4 + pALG11-316). Mature CPY (mCPY) is denoted by the
arrow. B, strains MCY1094 (ALG11),
NDY13.4 (alg11-1), and NDY13.4 harboring pALG11-316 were
streaked on YPAD or YPAD plates containing hygromycin B at 30 µg/ml
and incubated at 30 °C for 18 h.
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Fig. 3.
ALG11 encodes a conserved protein.
A, alignment of S. cerevisiae Alg11p-related
proteins. Proteins were identified using the BLAST (32) program and
aligned with DNASTAR (MegAlign) using the Clustal algorithm. The
GenBankTM accession number for each protein is as
follows: S. cerevisiae Alg11p, U12141; S. pombe Alg11p, CAA20913 (51); L. major, AAF77213;
D. melanogaster, AAF51756; C. elegans, P53993;
A. thaliana, AAD25934. B, alignment of S. cerevisiae Alg11p with Alg2-related proteins from S. cerevisiae and C. elegans. The GenBankTM
accession numbers for each of the proteins listed are as follows:
S. cerevisiae Alg2p, Z72587; C. elegans Alg2p,
U39649; S. cerevisiae Alg11p, U12141. C, proteins
were identified using the BLAST program (32), in which the 23-amino
acid sequence found to be conserved in Alg11p and Alg2p
(EHFGIAVVEAMACGTPVVAASGG) was used to search the data base, and
proteins were aligned as in panel A (note that only 20 of
more than 60 proteins containing this sequence are shown here). The
conserved consensus is indicated above the alignment.
GenBankTM accession numbers are as follows:
Acetobacter xylinum mannosyltransferase, X94981;
Anabaena HepB, U68035; Bacillus subtilis, L38424;
Hemophilus influenzae lipopolysaccharide biosynthesis
protein (LBP) Rd, U32842; Klebsiella
pneumococcus, D21242; Methanococcus jannaschii LPS,
U67601; Myobacterium leprae LPS, U00018; Pisum
sativum suc synthase, X98598; human PiGA, P37287; S. cerevisiae SPT14, Z73531x1; Salmonella choleraesuis
mannosyltransferase, X61917; Synechocystis
mannosyltransferase, D90901; Synechocystis LPS
galactosyltransferase, D63999; Xanthomas campestris GumH,
U22511; Y. enterocolitica trsE, Z47767.
1,6-linked mannose on
Man2GlcNAc2 to produce
Man3GlcNAc2 (12).
::URA3 colonies were grown at 37 °C (Fig. 4B). Thus, the ALG11 gene is
necessary for normal growth at room temperature and is essential for
growth at high temperatures. This severe phenotype is in stark contrast
to other glycosylation mutants that block lumenal ER steps in the
synthesis of the core OSL, suggesting a role for ALG11 at an
earlier stage.
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Fig. 4.
The ALG11 gene is essential
for growth at 37 °C. A, tetrad analysis of a diploid
strain heterozygous for the
alg11 ::URA3 deletion allele.
Diploids were sporulated, and dissected tetrads were analyzed for cell
viability at 25 °C. B, colonies were streaked onto YPAD
plates and incubated for 5 days at 25 °C or 37 °C.
alg11
double mutant was analyzed. The
alg3 mutant blocks the first mannose addition to the
Man5GlcNAc2-PP-Dol once flipped in to the ER
lumen yet accumulates Man5GlcNAc2 OSL (7, 14, 20). Isogenic haploid strains of opposite mating types but containing the alg11
::URA3 and
alg3
::HIS3 alleles were crossed, and
the heterozygous diploids were sporulated. Of the 40 tetrads dissected, none contained viable spores that were both His+ and
Ura+ (data not shown), demonstrating that the double
haploid mutant is lethal. These results are consistent with
ALG11 performing a function epistatic to ALG3,
which in combination so severely limits downstream lumenal ER OSL
glycan processing event(s) as to become synthetically lethal.
::URA3 strain rescued its
glycosylation defect, hygromycin B sensitivity, and growth phenotype
(data not shown), suggesting that the additional C-terminal HA
sequences do not alter the normal function of the Alg11 protein. The
ALG11-HA3 fusion was expressed constitutively
from the TPI promoter and could be detected by
immunoblotting cell protein extracts separated by SDS-PAGE (Fig.
5A). Although the expected molecular mass of Alg11-HAp is about 68 kDa, the tagged protein migrated as a 59-kDa species on denaturing gels, which may be explained
by its hydrophobicity. The protein is predicted to contain four
N-linked glycosylation sites, but none appear to be
utilized, because Alg11p was insensitive to digestion with endo H (Fig. 5A, compare lanes 3 and 4), whereas
Och1-HAp, a known glycosylated Golgi sugar transferase, was sensitive
to endo H (Fig. 5A, lanes 1 and
2).
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Fig. 5.
Western immunoblot and cytological analysis
of Alg11p. A, whole cell protein extracts were prepared from
yeast cells (SEY6210) expressing Och1-HA3p (lanes
1 and 2) or Alg11-HA3p (lanes 3 and 4). Proteins were incubated in the presence or absence
of endo H, fractionated by 8% SDS-PAGE, and subjected to Western blot
analysis using anti-HA antibodies. B, indirect
immunofluorescence of SEY6210 cells expressing HA3-tagged Alg11p. Fixed
cells were treated with anti-HA antibodies followed by FITC-conjugated
anti-mouse IgG and viewed by confocal microscopy.
OSL--
In
vivo labeling experiments employed [2-3H]Man or
[2-3H]Glc (see "Experimental Procedures"), and the
glycans released from OSL were chromatographed on Bio-Gel P-4 (Fig.
6). Panel A shows that the
steady-state [3H]Man-labeled OSL glycans (open
triangles) elute as Hex3GlcNAc2 (a), Hex6GlcNAc2 (b),
Hex7GlcNAc2 (c), and
Hex11GlcNAc2 (d). The central 85%
of each peak was pooled, and their structural identities were
investigated using endo H and
1,2-mannosidase digestion, as well as
methylation linkage analysis with the results summarized in Table
II. All glycans were
1,2-mannosidase-
and endo H-sensitive with the exception of
Hex3GlcNAc2. The lack of sensitivity to these
two glycosidases defines the Hex3GlcNAc2
polysaccharide as the core Man3GlcNAc2 (48),
and it was not investigated further. The endo H sensitivity of all
other glycans confirms the presence of the Alg3p-directed middle arm
1,3-linked Man (see Ref. 47 and Table II) and defines the minimum
structural component of this family of N-glycans as residues
2-5 and 7 (Scheme
IA).
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Fig. 6.
P-4 profile of in vivo
labeling of OSL and glycoprotein N-glycans in
alg11 yeast. A,
steady-state [3H]Man-labeled (
) or
[3H]Glc-labeled (
) N-glycans released from
OSL. The peaks are as follows: a,
[3H]Man3GlcNAc2; b,
[3H]Man6GlcNAc2; c,
[3H]Man7GlcNAc2; d,
[3H]Glc3Man8GlcNAc2
and
Glc3[3H]Man8GlcNAc2.
B, [3H]Glc-pulse-labeled N-glycans
released from cellular proteins (
). The peaks are as follows:
e, [3H]Glc1Man8GlcNAc;
f, [3H]Glc2Man8GlcNAc;
g, [3H]Glc3Man8GlcNAc.
C, steady-state [3H]Man-labeled (
)
N-glycans from cellular glycoproteins. The peaks are as
follows: h, [3H]Man6GlcNAc;
i, [3H]Man7GlcNAc; j,
Glc1-2[3H]Man7-8GlcNAc. The
internal raffinose standard centered at fraction number 184-186 is
shown in all panels (
). The elution positions of
Glc3[3H]Man9GlcNAc2,
[3H]Man9GlcNAc2,
[3H]Man7GlcNAc2, and
[3H]Man5GlcNAc2 released from
authentic wild-type OSL and the internal raffinose marker are shown
above relative to log10 of their hexose
size.
Characterization of alg11 N-glycans from OSL pulse-labeled for 2 min
with [2-3H]Man
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Scheme I.
1,2-mannosidase gave rise to two products on
the Bio-Gel P-4 that eluted as Hex5GlcNAc2 and
Hex4GlcNAc2 indicating the presence of isomers with either one or two Man
1,2-linked residues. In the
alg
9 background residue 6 is not added
to N-glycans because of the lack of Alg9p, the
presumptive
1,2-mannosyltransferase responsible for adding
residue 10, a required structural component for the addition of residue
6 (see Scheme IA and Refs. 6, 18, and 53). Once 6 is added,
residue 9 is the final
1,2-Man added to OSL. This strict order of
Man addition means that the Hex6GlcNAc2 containing one
1,2-Man must be missing both
1,2-linked residues 11 and 8, while having unsubstituted residue 6 and
1,2-Man 10. The
other Hex6GlcNAc2 from OSL with two
1,2-linked Man residues must contain residues 8 and 10 while missing
residue 6, because, as indicated above, the addition of 10 must precede
that of 9. The existence of both isomers implies that both
Man3GlcNAc2 and Man4GlcNAc2 OSLs are translocated into the ER
lumen to be further elongated by the Man-P-Dol-dependent
mannosyltransferases present in this compartment.
OSL (Fig. 6A, peak c),
hinting that this pool contained the rate-limiting dolichol-linked
biosynthetic intermediate in the alg11
background. Endo H
sensitivity, the loss of two
1,2-mannosidase-sensitive residues, and
the methylation pattern of the Hex7GlcNAc2 pool
(Table II) documents that Hex7GlcNAc2 contained
the above-defined core residues 2-5 and 7 plus the upper arm
1,6-Man residue 6 and only two of the three
1,2-linked Man residues 8, 9, and 10 shown in Scheme IC. Because residue 10 is required for the addition of 6 (see above), only two of the three possible Man7GlcNAc2 isomers would be present
in the OSL pool; the Man7 derived from elongation of the
Man3GlcNAc2-PP-Dol with
1,2-Man residues 9 and 10 (Scheme IB) and the Man7 derived from the
Man4GlcNAc2-PP-Dol with
1,2-Man residues 8 and 10 (see Scheme IC). Because
1,2-mannosidase removes
two residues from each, and their methylation patterns are the same, we
cannot specify the proportion of each in the pool.
1,2-mannosidase digestion (Table II). In [3H]Glc
pulse-labeling experiments (Fig. 6A, open
circles) a peak that eluted in the same position as the
[3H]Man-labeled Hex11GlcNAc2
showed the same endo H and
1,2-mannosidase sensitivities as the
[3H]Man-labeled form implying that they are the same
structure. The methylation analysis pattern generated by the
[3H]Man-labeled Hex11GlcNAc2 (see
Fig. 6, peak d and Table II) was consistent with this glycan
being identical to the authentic
Glc3Man9GlcNAc2 with its lower arm
shortened by the absence of residue 11 (see Scheme I, A and
C for comparison).
Glycoproteins--
Bio-Gel P-4 chromatographic
analysis of endo H-released glycans from [3H]Glc
pulse-labeled alg11
glycoproteins revealed the presence of Glc1-3Man8GlcNAc oligosaccharides (Fig.
6B, peaks e-g). On steady-state
[3H]Man labeling, Hex6GlcNAc,
Hex7GlcNAc, and Hex9GlcNAc were present in the
P-4 profile (Fig. 6C, peaks h-j).
1,2-Mannosidase and endo H sensitivities of the peaks from Fig.
6C, summarized in Table III,
are consistent with the following structural assignments: h,
[3H]Man6GlcNAc; i,
[3H]Man7GlcNAc; and j,
Glc1-2[3H]Man7-8GlcNAc.
Although
Glc1-2[3H]Man7-8GlcNAc and
[3H]Man7GlcNAc are the expected partially and
fully processed forms of
Glc3[3H]Man8GlcNAc2,
the presence of [3H]Man6GlcNAc in the endo
H-released glycans may appear inconsistent. However, if both
Man3GlcNAc2-PP-Dol and
Man4GlcNAc2-PP-Dol forms were translocated into
and elongated in the lumen of the ER as argued above, a
[3H]Man6GlcNAc would be expected on
alg11
glycoproteins, and this would be among the endo
H-released glycans. It would arise through Mns1p trimming of residue 10 from the [3H]Man7GlcNAc isomer defined in
Scheme IB. Another [3H]Man6GlcNAc
isomer on alg11
glycoproteins would be missing residue 9 but have residue 10 (Scheme IB) because of the inability of Msn1p to efficiently remove 10 in the absence of 9 (54). Both of these
Man6GlcNAc2 forms were identified on OSL in the
previous section and Table II and are shown to be present on
alg11
glycoproteins by the NMR structural studies of the
isolated glycans.
Characterization of steady-state [2-3H]Man-labeled N-glycans
from alg11 cellular glycoproteins
background, one from Man3GlcNAc2-PP-Dol and the
other from Man4GlcNAc2-PP-Dol. Processing of
these two glycan families would be quite different in the yeast ER and
Golgi. The first, leading to Man7GlcNAc in Scheme
IB with no Glc residues and without residue 8, would only be
processed in the ER by Mns1p's removal of residue 10 (54) and, quite likely, would not be expected to be a significant substrate for the early Golgi
1,6 branch-initiating enzyme Och1p (55, 56). The
only Golgi addition to this series would be
1,3-Man residues to 5, 7, 9, and/or residual residue 10 that escaped Mns1p activity (54). The
second series, containing Glc residues
G1, G2,
G3 (Scheme IC), and Man 10, would be
trimmed by Gls1p, Gls2p, and Mns1p. Because the series contains residue
8, it could act as a substrate, albeit weakly (56), for Och1p. Once
acted upon by Och1p, the series would, presumably, follow a normal
mannan elongation pattern (57).
Oligosaccharide Structures by
1H NMR--
To test the above predictions and determine
the steady-state distribution of N-linked glycans in
alg11
yeast, whole-cell glycoproteins were
deglycosylated, and the oligosaccharides were sized on Bio-Gel P-4
(Fig. 7). The profile reveals
Hex5-9GlcNAc sizes, which were collected,
rechromatographed on Bio-Gel P-4 (see "Experimental Procedures"),
and assigned as pools I-V, respectively. The Mr
of the final pools was confirmed by MALDI-TOF MS. Pools II and III were
solely Hex6GlcNAc and Hex7GlcNAc, respectively, whereas pools I, IV, and V had minor (<5% MS signal intensity) Hexx+1GlcNAc components. As described in an earlier study (58), pools I-V were subjected to Dionex high pH anion-exchange chromatography (data not shown), and the number of peaks and their areas generally agreed with the proportions of major and minor branch
isomers ultimately assigned in each pool. A fraction of the large V0 peak was collected as
alg11
mannan (Fig. 7, inset), and by MALDI-TOF
MS it was Hex30-49GlcNAc with an average size of
Hex39GlcNAc (Fig. 8,
inset).
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Fig. 7.
Preparative Bio-Gel P-4 chromatography of
N-linked glycans released from alg11
glycoproteins. Oligosaccharides were sized on the
2.6 × 67-cm Bio-Gel P-4 column, and 1.35-ml fractions were
collected and analyzed for neutral hexose by phenol-sulfuric acid assay
(
and
) and the presence of a
Man3GlcNAc[3H]ol internal marker by
scintillation spectrometry (
). Peaks representing separated
Hex5-9GlcNAc were collected as pools I-V and
rechromatographed on an analytical 1.6 × 96-cm Bio-Gel P-4 column
as described under "Experimental Procedures." Inset, a
portion of the V0 peak was collected as pool VI
and rechromatographed on the preparative column. The smallest glycans
of the V0 peak were collected as pool VI-I and
represent alg11
mannan.
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Fig. 8.
One- and two-dimensional DQF-COSY spectra at
500 MHz and 318 K of the C1- and C2-H region of alg11
mannan. The average size of the glycans
in pool VI-I by MALDI-TOF MS (Fig. 7, inset) was
Hex39GlcNAc, and the concentration in the NMR tube on the
basis of this size was 2 mM.
yeast, the structure of isomers present in each
pool were determined by 1H-NMR 1D and 2D DQF-COSY
experiments. The 1D spectra (not shown) were used for anomeric proton
chemical shift assignments and the integration of resonance intensities
as in previous studies (20, 53, 57-59). The apportionment of
intensities was assigned to structural isomers in each pool using
relevant C1-, C2-, and C3-H chemical shifts. As noted above for labeled
alg11
glycans (Table II and III), all structures deduced
have as their minimum core the Man4GlcNAc structure defined
in Scheme IB, consisting of residues 2
/
, 3-5, and 7, accompanied by their normal anomeric proton chemical shifts. Additions
of mannose in various linkages to this core diagnostically alters
anomeric and ring proton chemical shifts allowing structural assignment
on the basis of a large library of existing chemical shift data (5, 20,
53, 57-65). A summary of the altered N-glycosylation
pathway in alg11
yeast defined in the current work is
shown in Scheme II.
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Scheme II.
Mannan--
Essentially all of the structural isomers
assigned in the Hex5-9GlcNAc pools from
alg11
glycoproteins (Scheme II) are consistent with
Man3GlcNAc2-PP-Dol being translocated into the ER lumen and elongated maximally to the
Hex7GlcNAc2-PP-Dol, whose glycan structure is
shown in Scheme IB. However, the pulse and steady-state
labeling studies of both alg11
OSL and glycoprotein glycans described in the text, and Fig. 6 indicated that the
Hex8GlcNAc core shown in Scheme IC, which has
the single lower arm
1,2-linked Man (residue 8), was the principal
N-linked glycan intermediate. It seems unlikely that all of
the structures in Scheme II without residue 8 would be degradation
products of this Hex8 core intermediate (Scheme
IC). This could result if
Man3GlcNAc2-PP-Dol translocated into the ER
lumen led to this family of poorly elongated dead-end products that
accumulate on glycoproteins, whereas
Man4GlcNAc2-PP-Dol was more efficiently
processed to
Glc3Man8GlcNAc2-PP-Dol, transferred to protein, trimmed in the ER, and elongated in the Golgi to mannan (66).
1,2-Man 8, allowing Och1p to initiate outer
chain synthesis by adding
1,6-Man 12. Furthermore, we would expect
Mns1p to remove the middle arm
1,2-Man 10 from 7, because the mannan
chains whose origin was
Glc3Man8GlcNAc2-PP-Dol
contained residues 6 and 9, requisite for this activity (54). To test this prediction, pool VI, the more included portion of the
alg11
V0 peak in Fig. 7, was
isolated and rechromatographed on Bio-Gel P-4 (Fig. 7,
inset). Again, the most included portion, identified as pool
VI-I, was isolated and subjected to MS analysis and 1D DQF-COSY, 2D
DQF-COSY, and relayed ROESY 1H NMR experiments.
mannan (Fig. 8, inset). The Man anomeric
proton region of the 1D and 2D DQF-COSY 318 K spectra is
shown in Fig. 8. Core residues 3 (H1/H2 = 4.72/4.188 ppm) and 4 (H1/H2 = 4.862/4.131 ppm)
are present at 1 mol each. Note that the signal at
H1/H2 = 5.332/4.108 ppm for residue 5 when
substituted by
1,2-Man residue 8 (see Scheme IC). Residue 5 at this chemical shift integrates to more than 0.92 mol, indicating that nearly all of this mannan fraction was derived from the
Man4GlcNAc2-PP-Dol precursor. Essentially no
signal could be found for residual glucose in this glycan pool. In
addition, none of the anomeric proton signal of residue 7 is found at
5.403 ppm, where it resides when substituted by
1,2-Man 10 (67).
Thus, we conclude that the
Glc3Man8GlcNAc2 transferred to
alg11
glycoproteins is thoroughly processed by glucosidases I and II, as well as the ER
1,2-mannosidase, Mns1p, and
is the precursor of mannan in this mutant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is incompatible with the idea that Alg11p functions as a mannosyltransferase that catalyzes the addition of the fourth or
fifth mannose to OSL in the ER lumen. As expected for a protein that
functions in OSL synthesis, immunofluorescence analysis demonstrated that Alg11p is localized in the ER (Fig. 5). Epistasis analysis of an
alg11
alg3
double mutant demonstrates that
alg3 is not epistatic to alg11, strengthening the
notion that ALG11 does not simply function in the stepwise
ER lumenal assembly pathway. Rather, the mutant phenotype more closely
resembles that associated with mutants blocked in the early steps of
core biosynthesis that occur on the cytoplasmic face of the ER, and
indeed, analysis of lipid-linked oligosaccharides and those on
alg11 glycoproteins defines Alg11p to be involved in adding
the final
1,2-linked Man to the
Man5GlcNAc2-PP-Dol synthesized on the cytosolic
face of the ER. An assay to show that Alg11p is indeed an
1,2-mannosyltransferase remains to be developed.
1,2-Man residue so impairs the ER
translocation of the Man4GlcNAc2-PP-Dol that
Man3GlcNAc2-PP-Dol is also "flipped" into
the ER lumen. Normalizing the short N-glycans in pools I-V
derived from Man3GlcNAc2-PP-Dol and the
V0 mannan derived from
Man4GlcNAc2-PP-Dol (Fig. 7) for their
respective mannose content provides a rough estimate that nearly equal
amounts of both are translocated into the ER.
Man3,4GlcNAc2-PP-Dol both appear to be
elongated by the Man-P-Dol-dependent mannosyltransferases in the ER lumen to Man7,8GlcNAc2-PP-Dol. Of
particular interest is that the Man8 isomer missing
terminal
1,2-linked Man residue 11 (Scheme IC) is a
substrate for glucosylation (Fig. 6). Once transferred to protein,
Glc3Man8GlcNAc2 is a substrate for
ER removal of the three Glc residues by Gls1p and
Gls2p and the middle arm
1,2-Man 10 by Mnslp (54). This
Man7GlcNAc2 is a substrate for the Golgi
addition of the outer chain branch-initiating
1,6-Man 12 on core
residue 5 (68) by Ochlp (55). Indeed, residue 10 is completely absent
in alg11
mannan, and residues 8 and outer chain-initiating 12 are essentially quantitatively present (see Scheme ID, and Supplemental
Material, Fig. 1).
OSL indicates that once
Man4GlcNAc2-PP-Dol is formed it is translocated
and elongated to at least Man7GlcNAc2-PP-Dol, the next larger OSL that accumulates (see Structure
TI and Fig. 6A). As discussed
earlier, the proportion of the two possible isomers in the pool is not
known, but Man7B, the product of
Man3GlcNAc2-PP-Dol elongation, would be the
main candidate expected to accumulate prior to transfer to protein,
because it is not glucosylated. Accumulation of Man7A would
imply that addition of
1,2-Man 9 to 6 was rate-limiting relative to
the formation of
Glc3Man8GlcNAc2-PP-Dol, a small
amount of which accumulates on steady-state mannose and glucose
labeling (Fig. 6A).
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Structure I.
The first 1,2-Man, residue 8, is absent on all short
non-glucosylated oligosaccharides released from alg11
glycoproteins (Scheme II). These apparently accumulate as biosynthetic
end products because of their failure to be elongated significantly by
Ochlp (55) and serve in the Golgi as substrates for peripheral
1,2-Man elongation (69), as well as capping with
1,3-Man by Mnnlp
(70), and Man
1,3Man
1,3 elongation by additional
1,3-mannosyltransferases (71).
The Alg11 protein has been conserved through evolution. In addition to closely related proteins from other eukaryotes, Alg11p displays significant homology to Alg2p in S. cerevisiae and C. elegans. Mutants in alg2 are defective in an early step of the OSL assembly pathway, accumulating lipid-linked ManGlcNAc2 and Man2GlcNAc2 (7). ALG2 encodes a transferase that catalyzes mannose addition on the cytoplasmic face of the ER or a protein that regulates the activity of this transferase (12). Despite the sequence similarity between Alg11p and Alg2p, overexpression of ALG2 fails to rescue the alg11 mutation and vice versa (data not shown) suggesting that these two proteins do not perform overlapping functions.
A 23-amino acid sequence that is particularly well conserved in the
Alg2 and Alg11 proteins was observed. Strikingly, when this sequence
was used to search the data base, we found that it was present in over
60 proteins, most of which have known functions in carbohydrate
metabolism. These include different sucrose synthases (sucrose::UDP-glucosyltransferases) from a variety of
organisms, proteins from yeast, mice, and humans that function in the
synthesis of the glycosylphosphatidylinositol anchor, including
PigA and the yeast Spt14, and interestingly, a
large number of bacterial proteins that function in the biosynthesis of
the lipopolysaccharide (LPS) coat. The functional significance and
conservation of this motif to a family of glycosyltransferases was
previously recognized. A comparison of sequences common to a number of
bacterial -mannosyltransferases and their alignment by hydrophobic
cluster analysis identified several invariant residues. When this
sequence was used to search the data bases, it was found to exist in a
large family of proteins, including Alg2p (72), although Alg11p was not
identified in those studies. One feature common to all of these
proteins is the utilization of carbohydrates in the form of nucleotide
sugars. The conservation of this sequence among such a wide spectrum of proteins implicates this peptide as a domain involved in the binding of
nucleotide sugars or in the catalysis of their transfer, though not all
proteins that utilize nucleotide sugars contain this sequence. If
indeed this sequence reflects an active site domain that functions in
nucleotide sugar binding, it may provide a useful target for the
inactivation of these proteins, particularly in the case of the
bacterial proteins that synthesize LPS, as these are the surface molecules used by pathogens to colonize and survive in their hosts.
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ACKNOWLEDGEMENTS |
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We thank Niomi Peckham for early contributions to the ALG11 and OST4 projects. We thank Markus Aebi, Stephan te Heesan, and Maria Kukuruzinska for plasmids and members of the Dean laboratory for comments on the manuscript. We also gratefully acknowledge use of the Wadsworth biological mass spectrometry core facility and the Wadsworth structural NMR facility and thank Tracy Godfrey for help in preparing the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants GM48467 (to N. D.), GM33184 (to William J. Lennarz for the support of Q. Y.), and GM23900 (to R. B. T.) from the United States Public Health Service.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.
The on-line version of this article (available at
http://www.jbc.org) contains supplemental material.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U12141.
To whom correspondence should be addressed. Tel.:
631-632-9309; Fax: 631-632-8575; E-mail:
ndean@notes.cc.sunysb.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M010896200
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ABBREVIATIONS |
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The abbreviations used are:
ER, endoplasmic
reticulum;
OST, oligosaccharyltransferase;
OSL(s), oligosaccharide-lipid(s);
ORF(s), open reading frame(s);
CPY, carboxypeptidase Y;
PAGE, polyacrylamide gel electrophoresis;
endo H, endoglycosidase H and
endo--N-acetylglucosaminidase H (EC 3.2.1.96);
Dol, dolichol;
kb, kilobase pair;
HA, hemagglutinin;
TPI, triose
phosphate isomerase;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;
MALDI-TOF, matrix-assisted laser desorption ionization/time of flight;
1D, one-dimensional;
2D, two-dimensional;
MS, mass spectrometry;
LPS, lipopolysaccharide.
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