The yeast ALG11 gene specifies addition of the terminal 1,2-Man to the. cytosolic side of the endoplasmic reticulum.*

Transcription

1 JBC Papers in Press. Published on February 15, 2001 as Manuscript M MO: Print Version The yeast ALG11 gene specifies addition of the terminal 1,2-Man to the Man 5 GlcNAc 2 -PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum.* John F. Cipollo 1, Robert B. Trimble 1, Jung Hee Chi, 2 Qi Yan, 2 and Neta Dean 2* From the 1 Department of Biomedical Sciences, State University of New York at Albany, New York 12201, 1 The Wadsworth Center, New York State Department of Health, Albany, New York , and 2 The Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York at Stonybrook, New York Running Title: Role of ALG11 in oligosaccharide-lipid synthesis Key Words: N-glycosylation, yeast, ALG11, endoplasmic reticulum, oligosaccharide structures * To whom correspondence should be addressed. There is supplementary material in the online version of this article. Tel: (631) Fax: (631) ndean@notes.cc.sunysb.edu 1 Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

2 SUMMARY - 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 Man 5 GlcNAc 2 -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 Man 3 GlcNAc 2 -PPdolichol and Man 4 GlcNAc 2 -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 α1,2-linked Man residues. The former are elongated to mannan, while the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K., Nakanishi-Shindo, Y., Tonaka, A., Haga- Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, ) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1 kd 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 sixty proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif. 2

3 INTRODUCTION Asparagine-linked (N-linked) glycosylation is an essential and highly conserved modification that is required for the function of glycoproteins in eukaryotic cells. While 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 Glc 3 Man 9 GlcNAc 2 (for reviews see 1, 2). The assembly of the core oligosaccharide precursor occurs in two compartments. On the cytosolic face of the ER, Man 5 GlcNAc 2 is assembled stepwise from the nucleotide sugars UDP-GlcNAc and GDP-Man on to a lipid carrier, Dol-P 1 (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 Glc 3 Man 9 GlcNAc 2. 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 α1,2-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, S. cerevisiae, have been isolated that are defective in various steps involved in the synthesis of the Glc 3 Man 9 GlcNAc 2 core. Characterization of the alg (asparagine-linked glycosylation) mutants demonstrates that the early steps of core assembly are essential, while the later steps are not (7). Yeast with conditional mutations that interfere with the 3

4 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 Glc 3 Man 9 GlcNAc 2 is the preferred substrate (21, 24). While 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 identify the ALG11 lesion as a defect in the synthesis of the core lipid-linked oligosaccharide, specifically in the final step of Man 5 GlcNAc 2 -PP-Dol synthesis on the cytosolic face of the ER, addition of the terminal α1,2-linked Man. As a consequence of the elongation of Man 3,4 GlcNAc 2 -PP-Dol in the ER lumen by subsequent Man-P-Dol-dependent transferases, Man 7 GlcNAc 2 -PP-Dol 4

5 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. 5

6 EXPERIMENTAL PROCEDURES Yeast strains, media and genetic methods. Yeast strains were grown in either YPAD (1% yeast extract, 2% peptone, 2% dextrose, 50 mg/liter 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 (Boehringer Mannheim) 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 1. 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 medium lacking uracil. These transformants were replica plated on media containing 30 µg/ml hygromycin B. Plasmid DNA from hygromycin B resistant colonies was isolated, amplified in E. 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, 6

7 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 bp 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 subloned into prs316 (30) to produce palg and into Bluescript SK (Stratagene) to produce palg11- SK. The deletion plasmid, palg11 ::URA3, was constructed by replacing an internal 842 bp 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 BamH1/ClaI fragment from palg into prs306 (30). The plasmid was 7

8 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 palg into the URA3, 2µ plasmid, YEp24. An epitope-tagged version of ALG11, palg11-ha, was constructed by using PCR 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 residue. 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 triose phosphate isomerase (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 10 7 cells/ml and grown for three to four hours 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 (PMSF) to 0.5 mm, 1 mu of 8

9 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), were used at a 1:3000 dilution and detected by chemiluminescence (ECL, Amersham) 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 cells were grown overnight in YPD M KCl to a density of 4 x 10 7 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 (1x10 9 ) were resuspended in 200 µl YP + 0.1% glucose containing 250 µci of either [2-3 H]Glc (15 Ci/mmol) or [2-3 H]Man (20 Ci/mmol) purchased from American Radiolabeled Chemicals, St. Louis. The labeling and preparation of OSLs were performed as previously described (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 CHCl 3 /CH 3 OH (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 9

10 H then added to 50 mu/ml. The reactions were incubated at 37 o C for 16 h, and endo H activity was verified by hydrolysis of Man 6 GlcNAc 4 -Asn-dansyl, followed by paper chromatography of the released GlcNAc-Asn-dansyl moiety. The released glycans were solvent precipitated in 80% acetone as previously described (20). Chromatography. Oligosaccharides, released from alg11 OSL or glycoproteins (see below), were sized on a calibrated Bio-Gel P4 column (1.6 x 96 cm) eluted at room temperature with 0.1 N CH 3 COOH/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 alg11 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 prepared as described (20). Solid ammonium sulfate was added to the extract to 50% saturation, stirred for 30 min at 4 o C after the salt was in solution, and centrifuged at 30,000 x g for 15 min at 4 o C. The supernatant fraction (400 ml) was dialyzed over 72 h at 4 o C against three 6-L 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 dh 2 O and protein 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 mu/ml 10

11 overnight at 30 o C. Protein and glycans were precipitated at 20 o C by addition of four volumes of cold acetone, and the oligosaccharides 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 taken up in 1.5 ml 0.1N CH 3 COOH and initially chromatographed on a preparative Bio-Gel P-4 column (2.6 x 67 cm) with a typical profile shown in Fig. 7. Fractions (1.35 ml) corresponding to Hex 5,7,9 GlcNAc (odds) and Hex 6,8 GlcNAc (evens) from the two preparative columns were pooled separately and each rechromatographed into isolated glycan sizes on the calibrated 1.6 x 96 cm Bio- Gel P-4 column described above (not shown). The central 85% of the phenolsulfuric acid assay color for each peak was collected as Pools I-V corresponding to Hex 5-9 GlcNAc, 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/DMSO method. Briefly, the free hydroxyls of the oligosaccharides were deprotonated with NaOH/DMSO. Then CH 3 I was added to replace the free hydroxyls with methoxy groups. The methoxylated oligosaccharide was hydrolyzed in strong acid, evaporated under low pressure, 11

12 and applied to Whatman silica-gel 60A TLC plates. The plates were developed twice with MeCN-CHCl 3 -MeOH, 3:9:1 (V/V/V), thoroughly dried between each ascent, and rapidly dipped into a solution containing 3 g N-(naphthyl) ethylenediamine and 50 ml of concentrated H 2 SO 4 in 1 L of CH 3 OH. The plates were dried and placed in an oven for 10 min at 120 C. All saccharide standards were purchased from the Sigma Chemical Co. 1 H NMR Spectroscopy. Oligosaccharides ( pmol) were exchanged three times by rotary evaporation from 99.8% D 2 O and twice by lyophilization from 99.96% D 2 O. Lyophilized samples were dried over P 2 O 5 in vacuo for a day or more, then reconstituted in 0.5 ml % D 2 O (containing 1.35 mm acetone as an internal chemical shift reference) to final concentration of mm. Acetone protons were set at ppm and ppm for spectra taken at 300 K and 318 K, respectively. Samples were quickly transferred to 5 mm tubes (Wilmad Co., No. 535pp, previously washed and exchanged with 99.8% D 2 O), flame sealed, and examined at 300 K and 318 K by 1D and 2D DQF-COSY phase sensitive 1 H NMR spectroscopy at 500 MHz as previously described (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 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 ( 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 t 2 and 512 complex data points in the indirect t 1 dimension. The 2D relayed ROESY experiments were conducted at 12

13 318 o K as described by Cipollo et al. (45). 13

14 RESULTS The alg11 mutation affects the synthesis of N-linked glycoproteins. Resistance to sodium vanadate has previously been described 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 vs. 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 to that of the mature wild-type form (Fig. 1A). The mobility of CPY in alg11-1 was compared to 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 CPY s N-glycosylation, 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 due 14

15 to underglycosylation and not to proteolysis, and that the majority of CPY s oligosaccharides have the middle-arm α1,3-linked Man added to the Man 5 GlcNAc 2 -PP-Dol core OSL by Alg3p in the ER lumen, a requirement for endo H sensitivity (48). 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. Since 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 to 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). While 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 GPI 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 15

16 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 (ref. 27 and see 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 B s : hygromycin B r colonies and all of the hygromycin B r 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. 16

17 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 the data base (accession #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 databases (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 S. pombe (51), C. elegans, D. 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 α1,6-linked mannose on Man 2 GlcNAc 2 to produce Man 3 GlcNAc 2 (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 GPI anchor biosynthesis. This high degree of 17

18 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 Man 5 GlcNAc 2 -PP-Dol, respectively, and ALG6, which blocks addition of the first Glc to Man 9 GlcNAc 2 -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 Man 5 GlcNAc 2 -PP- Dol. To further investigate this mutant, the phenotype of an alg11 null allele was determined. An 832 bp 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 in Experimental Procedures. The disruption of one allele was confirmed by PCR 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 five days after the wild- 18

19 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 due to the alg11 mutation. Furthermore, no growth was observed when alg11 ::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. ALG3 is not epistatic to ALG11. As a further genetic test of whether ALG11 might function in the cytosolic assembly of Man 5 GlcNAc 2 -PP-Dol, the phenotype of an alg3 alg11 double mutant was analyzed. The alg3 mutant blocks the first mannose addition to the Man 5 GlcNAc 2 -PP-Dol once flipped in to the ER lumen, yet accumulates Man 5 GlcNAc 2 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. Alg11p is localized in the ER. To examine the physical properties of the Alg11 protein, an epitope-tagged ALG11 allele was constructed which encodes three 19

20 tandem copies of the HA epitope at the C-terminus. Expression of the Alg11- HAp fusion protein in an alg11 ::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-HA 3 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 weight 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, since Alg11p was insensitive to digestion with endo H (Fig. 5A, compare lanes 3 and 4), while Och1-HAp, a known glycosylated Golgi sugar transferase, was sensitive to endo H (Fig. 5A, lanes 1 and 2). 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 OSL. In vivo labeling experiments employed [2-3 H]Man or [2-3 H]Glc (Experimental Procedures), and the glycans released from OSL were chromatographed on Bio-Gel P-4 (Fig. 6). Panel A, shows the steady-state [ 3 H]Man labeled OSL glycans (open triangles) elute as 20

21 Hex 3 GlcNAc 2 (a), Hex 6 GlcNAc 2 (b), Hex 7 GlcNAc 2 (c), and Hex 11 GlcNAc 2 (d). The central 85% of each peak was pooled and their structural identities investigated using endo H and α1,2-mannosidase digestion, as well as methylation linkage analysis with the results summarized in Table II. All glycans were α1,2- mannosidase and endo H sensitive with the exception of Hex 3 GlcNAc 2. The lack of sensitivity to these two glycosidases defines the Hex 3 GlcNAc 2 polysaccharide as the core Man 3 GlcNAc 2 (48), and it was not investigated further. The endo H sensitivity of all other glycans confirms the presence of the Alg3p-directed middle-arm α1,3-linked Man (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). Digestion of Hex 6 GlcNAc 2 (Fig. 6A, peak b) with α1,2-mannosidase gave rise to two products on the Bio-Gel P-4, which eluted as Hex 5 GlcNAc 2 and Hex 4 GlcNAc 2 indicating the presence of isomers with either one or two Man α1,2-linked residues. In the alg 9 background residue 6 is not added to N- glycans due to the lack of Alg9p, the presumptive α1,2-mannosyltransferase responsible for adding residue 10, a required structural component for the addition of residue 6 (Scheme IA and refs. 6, 18, 53). Once 6 is added, residue 9 is the final α1,2-man added to OSL. This strict order of Man addition means that the Hex 6 GlcNAc 2 containing one α1,2-man must be missing both α1,2-linked residues 11 and 8, while having unsubstituted residue 6 and α1,2-man 10. The other Hex 6 GlcNAc 2 from OSL with two α1,2-linked Man residues must contain residues 8 and 10 while missing residue 6, since, as indicated above, the addition of 10 must precede that of 9. The existence of both isomers implies that both 21

22 Man 3 GlcNAc 2 and Man 4 GlcNAc 2 OSLs are translocated into the ER lumen to be further elongated by the Man-P-Dol dependent mannosyltransferases present in this compartment. The [ 3 H]Man-labeled Hex 7 GlcNAc 2 represented ~80% of the glycans released from pulse-labeled alg11 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 α1,2-mannosidasesensitive residues, and the methylation pattern of the Hex 7 GlcNAc 2 pool (Table II) documents that Hex 7 GlcNAc 2 contained the above-defined core residues 2-5 and 7 plus the upper-arm α1,6-man residue 6 and only two of the three α1,2- 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 Man 7 GlcNAc 2 isomers would be present in the OSL pool; the Man 7 derived from elongation of the Man 3 GlcNAc 2 -PP-Dol with α1,2-man residues 9 and 10 (Scheme IB) and the Man 7 derived from the Man 4 GlcNAc 2 -PP-Dol with α1,2-man residues 8 and 10 (see Scheme 1C). Because α1,2-mannosidase removes two residues from each and their methylation patterns are the same, we cannot specify the proportion of each in the pool. The Hex 11 GlcNAc 2 pool (Fig. 6A, peak d) lost the equivalent of two hexoses on α1,2-mannosidase digestion (Table II). In [ 3 H]Glc pulse labeling experiments (Fig. 6A, open circles) a peak that eluted in the same position as the [ 3 H]Man-labeled Hex 11 GlcNAc 2 showed the same endo H and α1,2-mannosidase sensitivities as the [ 3 H]Man-labeled form implying they are the same structure. The methylation analysis pattern generated by the [ 3 H]Man-labeled 22

23 Hex 11 GlcNAc 2 (Fig. 6, peak d, Table II) was consistent with this glycan being identical to the authentic Glc 3 Man 9 GlcNAc 2 with its lower arm shortened by the absence of residue 11 (see Scheme IA and C for comparison). 3 H-Glc- and 3 H-Man-labeled N-linked glycan structures on alg11 glycoproteins. Bio- Gel P-4 chromatographic analysis of endo H-released glycans from [ 3 H]Glc pulse-labeled alg11 glycoproteins revealed the presence of Glc 1-3 Man 8 GlcNAc oligosaccharides (Fig. 6B, peaks e-g). On steady-state [ 3 H]Man labeling, Hex 6 GlcNAc, Hex 7 GlcNAc, and Hex 9 GlcNAc were present in the P-4 profile (Fig. 6C, peaks h-j). α1,2-mannosidase and endo H sensitivities of the peaks from Fig. 6C, summarized in Table III, are consistent with the following structural assignments: h, [ 3 H]Man 6 GlcNAc, i, [ 3 H]Man 7 GlcNAc, and j, Glc 1-2 [ 3 H]Man 7-8GlcNAc. While Glc 1-2 [ 3 H]Man 7-8 GlcNAc and [ 3 H]Man 7 GlcNAc are the expected partially and fully processed forms of Glc 3 [ 3 H]Man 8 GlcNAc 2, the presence of [ 3 H]Man 6 GlcNAc in the endo H released glycans may appear inconsistent. However, if both Man 3 GlcNAc 2 -PP-Dol and Man 4 GlcNAc 2 -PP-Dol forms were translocated into and elongated in the lumen of the ER as argued above, a [ 3 H]Man 6 GlcNAc 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 [ 3 H]Man 7 GlcNAc isomer defined in Scheme IB. Another [ 3 H]Man 6 GlcNAc isomer on alg11 glycoproteins would be missing residue 9 but have residue 10 (Scheme IB) due to the inability of Msn1p to efficiently remove 10 in the absence of 9 (54). Both of these Man 6 GlcNAc 2 forms were identified on 23

24 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. The above experiments with labeled Glc and Man indicate that two series of truncated N-glycans arise in the alg11 background; one from Man 3 GlcNAc 2 - PP-Dol and the other from Man 4 GlcNAc 2 -PP-Dol. Processing of these two glycan families would be quite different in the yeast ER and Golgi. The first, leading to Man 7 GlcNAc 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 α1,6-branch initiating enzyme Och1p (55, 56). The only Golgi addition to this series would be α1,3-man residues to 5, 7, 9 and/or residual residue 10 that escaped Mns1p activity (54). The second series, containing Glc residues G 1, G 2, G 3, and Man 10, would be trimmed by Gls1p, Gls2p, and Mns1p. Since 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 alg11 oligosaccharide structures by 1 H NMR. In order 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 sized on Bio-Gel P-4 (Fig. 7). The profile reveals Hex 5-9 GlcNAc sizes, which were collected, rechromatographed on Bio-Gel P-4 (Experimental Procedures), and assigned as Pools I-V, respectively. The M r of the final pools was confirmed by MALDI-TOF MS. Pools II and III were solely Hex 6 GlcNAc 24

25 and Hex 7 GlcNAc, respectively, while Pools I, IV, and V had minor (<5% MS signal intensity) Hex x+1 GlcNAc components. As described in an earlier study (58), Pools I-V were subjected to Dionex HPAEC 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 V o peak was collected as alg11 mannan (Fig. 7, inset) and by MALDI-TOF MS was Hex GlcNAc with an average size of Hex 39 GlcNAc (Fig. 8, inset). To determine how N-glycans were processed in alg11 yeast, the structure of isomers present in each pool were determined by 1 H-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 done 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 Man 4 GlcNAc structure defined in Scheme IB, consisting of residues 2 α/β, 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, 57-64). A summary of the altered N-glycosylation pathway in alg11 yeast defined in the current work is shown in Scheme II. alg11 mannan. Essentially all of the structural isomers assigned in the 25

26 Hex 5-9 GlcNAc pools from alg11 glycoproteins (Scheme II) are consistent with Man 3 GlcNAc 2 -PP-Dol being translocated into the ER lumen and elongated maximally to the Hex 7 GlcNAc 2 -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 the Hex 8 GlcNAc core shown in Scheme IC, which has the single lower arm α1,2- 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 Hex 8 core intermediate (Scheme IC). This could result if Man 3 GlcNAc 2 -PP-Dol translocated into the ER lumen led to this family of poorly-elongated dead-end products that accumulate on glycoproteins, while Man 4 GlcNAc 2 -PP-Dol was more efficiently processed to Glc 3 Man 8 GlcNAc 2 -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 α1,2-man 8, allowing Och1p to initiate outer chain synthesis by adding α1,6-man 12. Furthermore, we would expect Mns1p to remove the middle-arm α1,2-man 10 from 7, since the mannan chains whose origin was Glc 3 Man 8 GlcNAc 2 -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 V o 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, 2D DQF COSY, and relayed ROESY 1 H NMR experiments. 26

27 MALDI-TOF MS provided a distribution of Hex GlcNAc with an average size of Hex 39 GlcNAc for the alg11 mannan (Fig. 8, inset). The Man anomeric proton region of the 1D and 2D DQF COSY 318 o K spectra is shown in Fig. 8. Core residues 3 (H 1 /H 2 = 4.72/4.188 ppm) and 4 (H 1 /H 2 = 4.862/4.131 ppm) are present at 1 mol each. Note the signal at H 1 /H 2 = 5.332/4.108 ppm for residue 5 when substituted by α1,2-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 Man 4 GlcNAc 2 -PP-Dol precursor. Essentially no signal could be found for residual glucose in this glycan pool. In addition, none of residue 7 s anomeric proton signal is found at ppm, where it resides when substituted by α1,2-man 10 (67). Thus, we conclude that the Glc 3 Man 8 GlcNAc 2 transferred to alg11 glycoproteins is thoroughly processed by glucosidases I and II, as well as the ER α1,2-mannosidase, Mns1p, and is the precursor of mannan in this mutant. 27

28 DISCUSSION 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 (26, 35). Sodium vanadate is a drug that enriches for yeast glycosylation mutants (27), although the molecular mechanism for this enrichment is not understood. The severely impaired growth phenotype associated with alg11 is incompatible with the idea that Alg11p functions as a mannosyltransferase that catalyzes the addition of the eighth or ninth 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 α1,2-linked Man to the Man 5 GlcNAc 2 -PP-Dol synthesized on the cytosolic face of the ER. An assay to show that Alg11p is indeed an α1,2-mannosyltransferase remains to be developed. The absence of the terminal α1,2-man residue so impairs the ER translocation of the Man 4 GlcNAc 2 -PP-Dol that Man 3 GlcNAc 2 -PP-Dol is also flipped into the ER lumen. Normalizing the short N-glycans in Pools I-V derived from Man 3 GlcNAc 2 -PP-Dol and the V o mannan derived from 28

29 Man 4 GlcNAc 2 -PP-Dol (Fig. 7) for their respective mannose content provides a rough estimate that nearly equal amounts of both are translocated into the ER. Man 3,4 GlcNAc 2 -PP-Dol both appear to be elongated by the Man-P-Dol dependent mannosyltransferases in the ER lumen to Man 7,8 GlcNAc 2 -PP-Dol. Of particular interest is that the Man 8 isomer missing terminal α1,2-linked Man residue 11 (Scheme IC) is a substrate for glucosylation (Fig. 6). Once transferred to protein, Glc 3 Man 8 GlcNAc 2 is a substrate for ER removal of the three Glc residues by Gls1p and Gls2p and the middle-arm α1,2-man 10 by Mnslp (54). This Man 7 GlcNAc 2 is a substrate for the Golgi addition of the outer chain branchinitiating α1,6-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 (Fig. 9, Scheme ID, and Fig. 1 in Supplemental Material). The accumulation of some Man 3 GlcNAc 2 -PP-Dol on steady-state labeling of alg11 OSL indicates that once Man 4 GlcNAc 2 -PP-Dol is formed it is translocated and elongated to at least Man 7 GlcNAc 2 -PP-Dol, the next larger OSL that accumulates (Fig. 6A). As discussed earlier, the proportion of the two possible isomers in the pool is not known, but Man 7 B, the product of Man 3 GlcNAc 2 -PP-Dol elongation, would be the main candidate expected to 29

30 accumulate prior to transfer to protein, since it is not glucosylated. Accumulation of Man 7 A would imply that addition of α 1,2-Man 9 to 6 was rate limiting relative to the formation of Glc 3 Man 8 GlcNAc 2 -PP-Dol, a small amount of which accumulates on steady state mannose and glucose labeling (Fig. 6A). The first α1,2-man, residue 8, is absent on all short non-glucosylated oligosaccharides released from alg11 glycoproteins (Scheme II). These apparently accumulate as biosynthetic end-products due to their failure to be elongated significantly by Ochlp (55) and serve in the Golgi as substrates for peripheral α1,2-man elongation (69) as well as capping with α1,3-man by Mnnlp (70), and Man α1,3man α 1,3-elongation by additional α 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 ManGlcNAc 2 and Man 2 GlcNAc 2 (7). ALG2 encodes a transferase that catalyses 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 sixty proteins, most of 30

31 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 GPI 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. 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. 31

32 FOOTNOTES 1. The abbreviations used are: Dol-P, dolichylphosphate; ER, endoplasmic reticulum; OST, oligosaccharyltransferase; OSL, oligosaccharide-lipid; ORF, open reading frame; CPY, carboxypeptidase Y; GPI, glycosylphosphatidylinositol; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; endo-h, endo-β-n-acetylglucosaminidase H (EC ); ManTase, mannosyltransferase. ACKNOWLEDGEMENTS We wish to thank Niomi Peckham for her 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 lab for comments on the manuscript. We also gratefully acknowledge use of the Wadsworth Biological Mass Spectrometry Core Facility, the Wadsworth Structural NMR Facility, and thank Ms. Tracy Godfrey for help in preparing the manuscript. 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 USPHS. 32

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38 FIGURE LEGENDS Figure 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 in 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 in Experimental Procedures. Proteins were separated by SDS-PAGE and subjected to immunoblot analysis with anti-cpy antibodies as in Panel A. The arrows denotes 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 in Experimental Procedures. The arrow denotes the mobility of chitinase. Figure 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 (NDY palg11-38

39 316). Mature CPY 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 hours. Figure 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 accession number for each protein is as follows: S. cerevisiae Alg11p (U12141); S. pombe Alg11p (CAA20913) (71); Leishmania major (AAF77213; D. melanogaster (AAF51756); C. elegans (P53993); Arabidopsis thaliana (AAD25934) B. Alignment of S. cerevisiae Alg11p with Alg2-related proteins from S. cerevisiae and C. elegans. The Genbank 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 twenty of more than sixty proteins containing this sequence are shown here). The conserved consensus is indicated above the alignment. Accession numbers are as follows: A. xylinum mannosyltransferase (X94981), Anabaena HepB (U68035), B. subtilis (L38424), H. influenza LSG (pir F64175), K. pneumococcus (D21242), M. jannaschii LPS (U67601), M. leprae LPS (U00018), P. sativum suc synthase (X98598), human PiGA (P37287), S. cerevisiae SPT14 (Z73531x1), S. choleraesuis ManTase (X61917), Synechocystis 39

40 ManTase (D90901), Synechocystis LPS galactosyltransferase (D63999), X. campestris GumH (U22511), Y. enterocolitica trse (Z47767). Figure 4. The ALG11 gene is essential for growth at 37. 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 five days at 25 C or 37 C. Figure 5. Western immunoblot and cytological analysis of Alg11p. A. Whole cell protein extracts were prepared from yeast cells (SEY6210) expressing Och1- HA 3 p (lanes 1 and 2) or Alg11-HA 3 p (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 antimouse IgG, and viewed by confocal microscopy. Figure 6. P-4 profile of in vivo labeling of OSL and glycoprotein N-glycans in alg11 yeast. A. Steady-state [ 3 H]Man-labeled ( ) or [ 3 H]Glc-labeled ( ) N- glycans released from OSL. The peaks are: a, [ 3 H]Man 3 GlcNAc 2 ; b, [ 3 H]Man 6 GlcNAc 2 ; c, [ 3 H]Man 7 GlcNAc 2 ; d, [ 3 H]Glc 3 Man 8 GlcNAc 2 and Glc 3 [ 3 H]Man 8 GlcNAc 2. B. [ 3 H]Glc-pulse labeled N-glycans released from cellular proteins ( ). The peaks are: e, [ 3 H]Glc 1 Man 8 GlcNAc; f, [ 3 H]Glc 2 Man 8 GlcNAc; g, 40

41 [ 3 H]Glc 3 Man 8 GlcNAc. C. Steady-state [ 3 H]Man-labeled ( ) N-glycans from cellular glycoproteins: h, [ 3 H]Man 6 GlcNAc; i, [ 3 H]Man 7 GlcNAc; j, Glc 1-2[ 3 H]Man 7-8 GlcNAc. The internal raffinose standard centered at fraction number is shown in all panels ( ). The elution positions of Glc 3 [ 3 H]Man 9 GlcNAc 2, [ 3 H]Man 9 GlcNAc 2, [ 3 H]Man 7 GlcNAc 2, and [ 3 H]Man 5 GlcNAc 2 released from authentic wild-type OSL and the internal raffinose marker are shown above relative to log 10 of their hexose size. Figure 7. Preparative Bio-Gel P-4 chromatography of N-linked glycans released from alg11 glycoproteins. Oligosaccharides were sized on the 2.6 x 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 the presence of a Man 3 GlcNAc[ 3 H]-ol internal marker by scintillation spectrometry ( ). Peaks representing separated Hex 5-9 GlcNAc were collected as Pools I-V and rechromatographed on an analytical 1.6 x 96 cm Bio-Gel P-4 column as described in the Experimental Procedures. Inset: A portion of the V o peak was collected as Pool VI and rechromatographed on the preparative column. The smallest glycans of the V o peak were collected as Pool VI-I and represent alg11 mannan. Figure 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 Hex 39 GlcNAc and the concentration in the NMR tube on the basis of this size was 2 mm. 41

42 A. ALG11 alg11-1 ost4-2 CPY B. ALG11 alg11-1 C. Endo H mcpy ALG11 alg11-1 vrg4-2 dgcpy chitinase Figure 1

43 A. ALG11 alg11-1 alg11-1 +palg11 mcpy B YPAD alg11-1 YPAD + hygromycin B alg11-1 alg palg11 ALG11 alg palg11 ALG11

44 A. ScAlg11p 48 S. pombe 31 C. elegans 25 Drosophila1 23 Arabidopsis 22 Leishmania 151 S L NPFSKKSSLLNRAVASC - -GEKNVKVFG F F H P Y C N A G G G G EKV L WKA VD I TLRKDAK - R L I KK I APVKASLYKQVGV - - EPKLARTVG F F H P Y C N A G G G G E R V L WTA VKSVQ TEFPN - A L I I P F S L Y S G F R R K S K T V A F F H P Y C N A G G G G E R V L WAA I R T M Q KKFPD - L F L R QW L L G R K N K L H T S S E N G I N V G I F H P Y C N A G G G G E R V L WC A VRALQEKYQN- F L S V I N A R K S R K R A V G F F H P Y T N D G G G G E R V L WC A VKA IQ EENPD- R MLTFTVLVLAALALLCHCRKRAYARNTVG F L H A AAGAGGGGE R V L W V A LDGLQHADAAR ScAlg11p 105 S. pombe 88 C. elegans 74 Drosophila1 77 Arabidopsis 67 Leishmania N V I V I Y S G DFVNGE NVTPEN I LNNVKAKF DYDL D SDR- I FF I S L K L R YLVDSS V I S V V Y T G D N V S KAE I LRRVKNTF E I D L D SSK- I VF VYL K L R FLVSAT H K Y FVY S G D T D A TKEQ I L L KARQRFG I E L D PSN- IQF I Y L HWR TLVEAR A R M V I Y T G D I D A S PNS I LQKAKNVFN I A V D SDN- VKF VFLKQR HW I E A K L D C V I F T G D H D S S SDSLARRAVDRFGVHLQS P - - P K V I H L NKRK W I E E S GVKRQYV L F T N EYKPADRLSAESSDQH L L S LVEKQFS I R L L RPVRF I YLRPALTRWLSGD ScAlg11p 157 S. pombe 135 C. elegans 122 Drosophila1 125 Arabidopsis 114 Leishmania 271 TWK H F T L I G QA IG - - S M I L A F E S I TWH R F T L L G QSLG - - S M I L GFEA I Y R F A P D H Y K H C T M L F Q ALA - - GL I L A L E AWF RMV P A N Y P H F T L L G QS IG - - S MVVG LE ALCRFPPD T Y P H F T M I G QSLG - - S VYL AWE ALRMFTPL A Y P RLT L L L Q TFWGG AALFYEV A VANAVTP I Q C P P D I W I D T M G YPFS Y P I I A R F L R R I P I V T Y T H Y I F I D T M G YAF T FCVVKSFQN- I PV GAY V H Y VF I D S M G YPLSLPA F R LSG- - SKV VAY V H Y I Y I D T M G YAF T Y PLFRYLAQ-SKVGC Y V H Y YFLDTSGYAF T Y PLAR I F G - - C K V VCY T H Y I VVE T V G VPFA Y P L L R L L A G - C M V I S Y T H Y ScAlg11p 215 S. pombe 192 C. elegans 178 Drosophila1 182 Arabidopsis 170 Leishmania 330 ScAlg11p 265 S. pombe 239 C. elegans 238 Drosophila1 242 Arabidopsis 215 Leishmania 390 ScAlg11p 306 S. pombe 276 C. elegans 280 Drosophila1 280 Arabidopsis 275 Leishmania 427 ScAlg11p 362 S. pombe 330 C. elegans 336 Drosophila1 339 Arabidopsis 335 Leishmania 480 ScAlg11p 422 S. pombe 388 C. elegans 394 Drosophila1 397 Arabidopsis 393 Leishmania 538 P IMSKDML N K L F K M P K K G I K V Y G K I L Y W K V F ML I Y QS IG S K I D I V I T N ST P T I STDML K S L K Q V S L L A K V K M A Y W R W F AQ LY SDAGSHADYVM T N S S P T I SCDM LDVVESRQETFNNSST I A QSNVL SWGKLTYYRLFAC LY WLAGKAAHVGMV N G S P V I STDMLKRVQQRQMSHNN KKYVA RNP FL TWTKLAYYRLFSRMY KWVGCCAE T IM V N S S P T I SLDM I S R V RQRNSMY NN DAS I A K S WM Y GMVG SC THLAM V N S S P I V S S A M TQRV RSGEVSHTNSPTVAWNPML RCAKVVYYGVFSLLYRCMG FFPNVVL T N S S W TNNH I K Q I W QS - NTCKI I Y P P CSTEKLVD W K Q K F G T A K G E R W TRNH I A S L W GKD IQLSVV F P P C N T S E L E K I D I N R K R W TQR H I T S I W SR- RDVS I V Y P P CDVEAFLN- - W TENH I L Q L W DVP FKTHRV Y P P C E V S I ESVAESLLEDTK W TKS H I E V L W R I PER I TRV Y P P CDTSGLQV W T Q N H VQS I FWP - R AC I R L Y P P C D V A G H L K S L Q H T E K G D I TFRQLESYDFLFAD I LGLLQAFPLERSSD F A A G S Q P P A L R LNQA IVLAQ F R P E K R H K L I I E S F ATFLKNL PDSVSP I K L I M A G STRSKQDENYVKS EPTLLYLAQYRPEK N H E NV L RS F ALYFEQHPDSP - - AK L L L V G SVRGEEDMC F V NH TVRL LS VGQ I R P E K N H K L Q L EVLHDVKEPL EKMGYNVE L C I A G GC R NEEDQERVKM EF I I LS VGQ F R P E K D - H P L QLQA I Y E L R T L LAQDEALWNQ I K LV IVG SCR NEDD YER LKN PPKI I S VAQ F R P E KVRDPAHM L Q L EAFSLALEKL DADVPRPK LQFVG SCR NNSD EER LQK NNR I VS V G Q F R P E K N H M L Q L VAFHAAMPR L PRDA K L VM I G GARNADDRKR AEQ L QDWSENVLKI PKHL I SF EKN L P FDKI E I L L NKS TFG VNAMWNE H F G I A V V E Y M ASG L I P L KTLA TEL N - - L Q S K V K F VVDAPWPKVVEY L GTCS I GVNYMWNE H F G IGVVEYMAAGL I P L KNEA EKL D - - I S E Q L I WQ L N V P YEDLVVE L SKAL I S I H T M H N E H F G I S V V E A M A A S T I I MQ D L TKHL S - - L E N N V Q F SVNV P YEDLLKLYQ TAH I G I H T M W N E H F G IG I V ESMA A GL I M L KDRA VEL K - - V D G D V Q F YKNAMYRE LVE L L GNAVAG L H G M I D E H F G I S V V E Y M A A GA I P L HVRA KELG - - I E E Q V E L L V N ATVAEVQAE L GKCV I G L H T M RDE H F G I V L L E Y L A A G C I P I V H A S A G P L L D I VTPWDANGNIGKA PPQWE VVNNS G G PKFD I V I P W I G K P T G FHA S T I S E LSNDS G G PRMD I VKDYEG HC VG Y L S I T KEE VAH K S G G P L L D I VETSAG SQNG F L ATDAVE I A H N S A G PKMD I VLEEDGQKTG F L AETVEE LGH R S G G VELD I LNSPD- - - L G F L A V T AEE LQKKYFAKLEDDGETTGFFFKEPSDPDYNT Y A EAYHKALTLSPQEQLEMR I NA RSACARF Y V E T I L K I VEEGLKKRNDTRKYA RKS L TRF Y A EN I LN I I V N - N S E M N G I R N A A R Y A EA I LE I VKMNETERLKMAESA RKRAARF Y A AAMVE I CEMRLRDPDRYVQFQKRGSE - - ScAlg11p 482 S. pombe 448 C. elegans 454 Drosophila1 0 Arabidopsis 453 Leishmania 593 TKDPLRYPNLSDLF L Q I T K L D Y D C L R V M G - GEHVFMRD FG - NV F A K L L R E D Y S R T GEAAF EVNSLEQRNRKSAVN I FSFKL I I FS SEQRFC E D F K T A I R P I F T G P L K HVKS F DDSS FRTRF VELVSEYVYAC Figure 3A

45 B. 1 MGSAWTNYNFEE-VKSHFGFKKYVVS---SLVLVY GLIKVLTWIFRQWVYSSLNPFSKKSSLLNRAVASCGEKNVKVFGFF HPY sc alg11 1 MIEKDKR--SIAFIHPDL----GIGG---AERLVV DA ALGLQ QQGHSVIIY TSH sc alg2 1 MFSELKRYSVLSLYTCAL----FFSV---PLSLVIWFVRRELCTET SVAGE KQMHVVIVHPEQWNGGSDR ce alg2 81 CN AGGGGEKVLWKAVDITLRKDAKNVIV IYSGDFVNGENVTPENILNNVKAKFDYDLDS- sc alg11 46 CD KS HCFE--EVKNGQLKVE VYG DFLPT sc alg2 64 CTVALIRHFVSQGHRVTWLTTMIDEYWKN HTFDGVEIREVGLKLHPGDWWSQNVALGWHMVFS NLNP- ce alg DRIFFISLKLRYLVDSSTWKHFTLIGQAIGSMILAFES---IIQCPPDIWIDTMGYPFSYPII-ARFLR sc alg11 72 NFLGRFFIVFATIRQLYLVIQLILQKKVNAYQLI IIDQLSTCIPL LH-I--FS sc alg DVA IIDHSASCVPM IKWR--FP ce alg2 205 RIPIVTYTHYPIMSKDML NKLFKMPKKGIKVYGKILYWKVFMLIYQSIGSKIDIVITNSTWTNNH----IKQIWQSNT-CKIIYPPCSTE sc alg SATLMFYCHFPD---QLL AQRAGLLKK IYRLPFDLIEQFSVSAADTVVVNSNFTKNTFHQTFKYL--SND-PDVIYPCVDLS sc alg2 151 QCKILFYCHFPQ---QLV TPSRFFLYR WYAKLIGIVEEELFGHIDQIFVNSNFTATQFCKVMPNI--EKNKVRVVYPPCDID ce alg2 290 KL-VDWKQKFGTAKGERLNQA IVLAQFRPEKRHKLIIESFA-TFLKNLPDSVSPIKLI------MAGSTRS KQDENYV sc alg TIEIE----DIDKKFFKTVFNEGDRF-YLSINRF--EKKKDVALAINGFALSED--QINDNVKLVICGGYDERVAENVEYLKELQSLADEYELSHTTIYY sc alg2 228 WI-VS----ASERPVSRAQRAKNETYTFLSMNRFWPEKRLDIIIEAAS-ILKQK--GYHFHVQLA------GSVMPHI PESRIYY ce alg2 360 KSLQDWSE--NVLKIPKHLISFEKNLPFDKI-EILLNKSTFGVNAMWNEHFGIAVVEYMASGLIPIVHASAGPLLDIVTPWDAN GNI sc alg QEIKR-VSDLESFKANNSKII-FLTSISSSLKELLLERTEMLLYTPAYEHFGIVPLEAMKLGKPVLAVNNGGPL-ETIKSYVAG ENE sc alg2 299 EVLQR-MT--EELNVTD-MVT-FIPSPTDKVKFQLYQQCDTALYTPPNEHFGIVPIEALDQRRPVIVCDSGGPA-ETVLEDITG TKI ce alg2 444 GK------APP--QWELQKKY FAKL sc alg SS------ATGWLK-PAVPIQ WATAIDESRKILQNGSVN---FERNGPLRVKKY sc alg2 380 AKPCGELLAEAMLH-HMNKRD WPELDTDE G---YAKQ-RHRLETE ce alg EDDGETTGF FFKEPSDPDY NTTK DP LRYPNLSDL-- sc alg FSREAMTQSF EENVEK VIWKEKKYYPWEI F------GI-- sc alg FSTRGFCGNI DRAIAE MM GT L------EI-- ce alg FLQ-ITKLDYDCL-RVMGARN QQYSLYKFS----DLKFDKD WEN F sc alg SFSNFILHIA--F-IKILPNNPWPSYLWPLLWYYILRTTYGEFTGHLYSLSPTLM-KKYN sc alg SSS-EPTTTP--L-VDTIVHQ PTATEVYSK----P-QQYN ce alg2 534 VLNPIC KLLEEEER G sc alg VYLNKQCIPPEEKIIYIGEEISK CK sc alg KAAHSRRA QA ce alg2 C. -.E.FGL..VEAMA.G.PV...GG EGFGLVVVEAMACGLPVVAT--GG WNEHFGIAVVEYMASGLIPIVHASAGPLLDIVTPWD --EHFGISVVEAMAASTIILSNDSGGP -YEHFGIVPLEAMKLGKPVLAVNNGGPLETI -NEHFGIVPIEALDQRRPVIVCDSGGPAE --EGFGLAAVEAMSAGLVPIL -FEGFGLAITESLACGTPVLCTPIG --ESFGLVLLEAMACGVPCIGTNIG --EGLPLVVIEAMAFGLPIVAFNCSP -WESFGLVAVEAQLYGVPVI -YEPFGIVALEAMAAGTPVVVSSVGGLMEII --ESFGLVAVEAQACGTPVVAAAVG -YEAFGLTVVEAMATGLPTFATLNGGPAEII -TEAFCMAIVEAASCGLQVVSTRVGGIPEV -YEHFGIVPLEAMKLGKPVLAVNNGGPLETI -TEAFGTILVEAASCNLLIVTTQVGGIPEV -YEGFGLPVVEGMACGIPVLTS LYEGFGLPVVEGMACGIPVL -YEGFGLPVVEAMNFGLPIITAGAGS----L LKVVEYMAAGLPIVA-SCIG --EGFGIAAVEAMSAGLIPIL W-EGFPLVITEAMACKKIIVATD-AG Consensus Majority S. cer Alg11 C. eleg Alg11 S. cer Alg2 C. eleg Alg2 A. xylinum ManTase Anabaena HepB B. subtilis H. influenza LSG K. pneumococcus M. jannaschii LPS Myco leprae LPS P.sativum suc synthase PIGA human S. cer ORF YGLo65c S. cer SPT14 S. choleraesuis Mtase S. typhimurium Mtase Synechocystis Mtase B Synechocystis SPS LPS GTASE X. campestris GumH Y.enterocolitica trse Figure 3

46 A. B. 25 C 37 C alg11d::ura3 ALG11 alg11d::ura3 ALG11 Figure 4

47 A. endoh Och1-HA Alg11-HA B. Alg11-HA ER Figure 5