The Yeast ALG11 Gene Specifies Addition of the Terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-Glycosylation Intermediate Formed on the Cytosolic Side of the Endoplasmic Reticulum*,

John F. CipolloDagger §, Robert B. TrimbleDagger §, Jung Hee Chi, Qi Yan, and Neta Dean||

From the Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1,2-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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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).

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 alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table I
Yeast strains used in the present study

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- (Stratagene) to produce pALG11-SK-.

The deletion plasmid, palg11Delta ::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.

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-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.

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 alg11Delta 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).

Chromatography-- Oligosaccharides, released from alg11Delta 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).

Isolation of Oligosaccharides from alg11Delta 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."

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 alpha 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.

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).


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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.

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.



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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.

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 alpha 1,6-linked mannose on Man2GlcNAc2 to produce Man3GlcNAc2 (12).

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 alg11Delta ::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 alg11Delta ::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.

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 alg3Delta alg11Delta 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 alg11Delta ::URA3 and alg3Delta ::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.

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 alg11Delta ::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.

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 alg11Delta 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 alpha 1,2-mannosidase digestion, as well as methylation linkage analysis with the results summarized in Table II. All glycans were alpha 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 alpha 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 alg11Delta yeast. A, steady-state [3H]Man-labeled (triangle ) or [3H]Glc-labeled (open circle ) 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 (black-triangle) 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 (black-square). 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.

                              
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Table II
Characterization of alg11Delta N-glycans from OSL pulse-labeled for 2 min with [2-3H]Man


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Scheme I.  

Digestion of Hex6GlcNAc2 (Fig. 6A, peak b) with alpha 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 alpha 1,2-linked residues. In the algDelta 9 background residue 6 is not added to N-glycans because of the lack of Alg9p, the presumptive alpha 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 alpha 1,2-Man added to OSL. This strict order of Man addition means that the Hex6GlcNAc2 containing one alpha 1,2-Man must be missing both alpha 1,2-linked residues 11 and 8, while having unsubstituted residue 6 and alpha 1,2-Man 10. The other Hex6GlcNAc2 from OSL with two alpha 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.

The [3H]Man-labeled Hex7GlcNAc2 represented ~80% of the glycans released from pulse-labeled alg11Delta OSL (Fig. 6A, peak c), hinting that this pool contained the rate-limiting dolichol-linked biosynthetic intermediate in the alg11Delta background. Endo H sensitivity, the loss of two alpha 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 alpha 1,6-Man residue 6 and only two of the three alpha 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 alpha 1,2-Man residues 9 and 10 (Scheme IB) and the Man7 derived from the Man4GlcNAc2-PP-Dol with alpha 1,2-Man residues 8 and 10 (see Scheme IC). Because alpha 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.

The Hex11GlcNAc2 pool (Fig. 6A, peak d) lost the equivalent of two hexoses on alpha 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 alpha 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).

3H-Glc- and 3H-Man-labeled N-Linked Glycan Structures on alg11Delta Glycoproteins-- Bio-Gel P-4 chromatographic analysis of endo H-released glycans from [3H]Glc pulse-labeled alg11Delta 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). alpha 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 alg11Delta 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 alg11Delta 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 alg11Delta glycoproteins by the NMR structural studies of the isolated glycans.

                              
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Table III
Characterization of steady-state [2-3H]Man-labeled N-glycans from alg11Delta cellular glycoproteins

The above experiments with labeled Glc and Man indicate that two series of truncated N-glycans arise in the alg11Delta 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 alpha 1,6 branch-initiating enzyme Och1p (55, 56). The only Golgi addition to this series would be alpha 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).

Assigning alg11Delta Oligosaccharide Structures by 1H NMR-- To test the above predictions and determine the steady-state distribution of N-linked glycans in alg11Delta 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 alg11Delta 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 alg11Delta 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 (black-diamond  and black-triangle) 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 alg11Delta 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 alg11Delta 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.

To determine how N-glycans were processed in alg11Delta 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 alg11Delta glycans (Table II and III), all structures deduced have as their minimum core the Man4GlcNAc structure defined in Scheme IB, consisting of residues alpha /beta , 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 alg11Delta yeast defined in the current work is shown in Scheme II.


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Scheme II.  

alg11Delta Mannan-- Essentially all of the structural isomers assigned in the Hex5-9GlcNAc pools from alg11Delta 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 alg11Delta 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 alpha 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).

If this occurred, we would expect to find essentially all of residue 5 substituted by alpha 1,2-Man 8, allowing Och1p to initiate outer chain synthesis by adding alpha 1,6-Man 12. Furthermore, we would expect Mns1p to remove the middle arm alpha 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 alg11Delta 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.

MALDI-TOF MS provided a distribution of Hex30-49GlcNAc with an average size of Hex39GlcNAc for the alg11Delta 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 alpha 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 alpha 1,2-Man 10 (67). Thus, we conclude that the Glc3Man8GlcNAc2 transferred to alg11Delta glycoproteins is thoroughly processed by glucosidases I and II, as well as the ER alpha 1,2-mannosidase, Mns1p, and is the precursor of mannan in this mutant.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alg11Delta 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 alg11Delta alg3Delta 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 alpha 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 alpha 1,2-mannosyltransferase remains to be developed.

The absence of the terminal alpha 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 alpha 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 alpha 1,2-Man 10 by Mnslp (54). This Man7GlcNAc2 is a substrate for the Golgi addition of the outer chain branch-initiating alpha 1,6-Man 12 on core residue 5 (68) by Ochlp (55). Indeed, residue 10 is completely absent in alg11Delta mannan, and residues 8 and outer chain-initiating 12 are essentially quantitatively present (see Scheme ID, and Supplemental Material, Fig. 1).

The accumulation of some Man3GlcNAc2-PP-Dol on steady-state labeling of alg11Delta 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 alpha 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 alpha 1,2-Man, residue 8, is absent on all short non-glucosylated oligosaccharides released from alg11Delta 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 alpha 1,2-Man elongation (69), as well as capping with alpha 1,3-Man by Mnnlp (70), and Man alpha 1,3Man alpha 1,3 elongation by additional alpha 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 alpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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-beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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