John F. Cipollo 1,3 and Robert B. Trimble 2,3,4

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1 Glycobiology vol. 12 no. 11 pp , 2002 The Saccharomyces cerevisiae alg12 mutant reveals a role for the middle-arm α1,2manand upper-arm α1,2manα1,6man- residues of Glc 3 Man 9 GlcNAc 2 -PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus John F. Cipollo 1,3 and Robert B. Trimble 2,3,4 3 Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, NY 12201, USA and 4 Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201, USA Received on April 12, 2002; revised on June 20, 2002; accepted on July 5, 2002 N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc 3 Man 9 GlcNAc 2 from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man 7 GlcNAc 2 -PP-Dol intermediate. We show that the Man 7 GlcNAc 2 released from alg12 -secreted invertase is Manα1,2Manα1,2Manα1,3(Manα1,2Manα1,3Manα1,6)- Manβ1,4-GlcNAcβ1-4GlcNAcα/β, confirming that the Man 7 GlcNAc 2 is the product of the middle-arm terminal α1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12 and wild-type cells, the central-arm α1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12 background. This confirms in vivo earlier in vitro experiments showing that the upperarm Manα1,2Manα1,6-disaccharide moiety, missing in alg12 Man 7 GlcNAc 2, is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-golgi α1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A. Key words: glycan 1 H NMR/glycoprotein processing/n-linked oligosaccharides/s. cerevisiae/yeast alg mutants Introduction With the exception of some protists, all eukaryotes transfer Glc 3 Man 9 GlcNAc 2 from dolichol pyrophosphate (PP-Dol) to specific Asn residues in nascent polypeptides included in 1 Present address: Boston University Goldman School of Dental Medicine, Boston, MA , USA 2 To whom correspondence should be addressed; trimble@wadsworth.org AsnXaa Ser/Thr sequons, where Xaa is any amino acid except Pro. Saccharomyces cerevisiae hasbecomeauseful model system for glycosylation studies because this yeast is easily adapted to genetic and molecular techniques and its genome has recently been completely sequenced (Cherry et al., 1997). N-linked glycosylation in S. cerevisiae has been characterized by isolating and studying members of the alg (Burda and Aebi, 1999), mnn (Ballou, 1990), and ktr (Lussier et al., 1999) families of mutants. Many of the steps in yeast N-glycosylation show a high degree of pathway conservation with higher eukaryotes, including steps in oligosaccharide lipid (OSL) synthesis, glycan transfer to nascent proteins by oligosaccharyltransferase (OST), and removal in the endoplasmic reticulum (ER) of the glucotriose moeity and the central-arm α1,2-linked Man from the newly transferred Glc 3 Man 9 GlcNAc 2 (Trimble et al., 1980; Byrd et al., 1982). Mutants with lesions late in the yeast OSL pathway display no selectable phenotype. However, recently, Aebi and co-workers isolated the late-acting genes ALG3, ALG6, ALG8, ALG9, and ALG10 by rescuing through complementation their respective synthetically lethal phenotypes occurring in conjunction with mutations in subunits of OST (Stagljar et al., 1994; Aebi et al., 1996; Burda et al., 1996; Burda and Aebi, 1998; Reiss et al., 1996). This screening technique, in addition to 3 H-Man suicide (Huffaker and Robbins, 1982, 1983) and sodium vanadate resistance (Dean, 1995), have identified a mutant in nearly everystepinglc 3 Man 9 GlcNAc 2 -PP-Dol synthesis (Scheme 1A) and its subsequent processing to mannan. The remaining unidentified N-glycan pathway genes, not found by the more conventional methodologies, are now being isolated by applying homology-searching algorithms to the annotated S. cerevisiae genome (Cherry et al., 1997). ALG12 (ECM39) was first identified by transposon-distruption/plasmid rescue methodology and assigned a role in cell wall formation, because on mutation, ecm39 yeast showed growth defects in the presence of papulacandin, a glycolipid that hinders β1,3-glucan synthesis (Lussier et al., 1997). However, the actual function of the protein encoded by ECM39 was not determined. ALG12 was identified independently using a database searching algorithm with Alg9p as query sequence (Burda et al., 1999), and an alg12, wbp1-2 double mutant showed no discernible phenotype beyond that of the wbp1-2 parent, demonstrating the mild nature of the alg12 glycosylation defect. The alg12 mutant accumulates Man 7 GlcNAc 2 - PP-Dol, a small amount of Glc 3 Man 7 GlcNAc 2 -PP-Dol, and reveals some hypoglycosylation of vacuolar carboxypeptidase Y (CPY) (Burda et al., 1999). 749

2 J.F. Cipollo and R.B. Trimble Scheme 1. Representative glycan structures and their anomeric 1 H NMR resonances (ppm). (A)TheGlc 3 Man 9 GlcNAc 2 transferred to protein in wild-type cells. The boxed area indicates the residues conserved in the core alg12 glycan; (B) alg9 Man 6 GlcNAc 2 ;(C) alg12 Man 7 GlcNAc 2 ;(D)thealg12 glycan containing all Golgi modifications seen in this study. The Man added by Och1p is boxed (Nakayama et al., 1997). Recently, we showed in alg9 yeast that the central-arm α1,3-linked Man residue 7 added by Alg3p (Scheme 1) potentiates the Alg6p, Alg8p, and Alg10p glycosyltransferases, the Gls1p/Cwh41p and Gls2p glucosidases I and II, and the Golgi Ochlp initiation of α1,6-man outer chain (Cipollo and Trimble, 2000). Here, we extend our understanding of the role individual Man residues in OSL play in subsequent N-glycan processing by defining the structure of alg12 N-glycans and the fate of α1,2-linked Man residue 10 added by Alg9p (Scheme 1A). Interestingly, invertase and protease A (PrA) are hypoglycosylated in alg12, leading to their instability, which provides a practical indication of the role proper synthesis and processing of N-glycans plays in glycoprotein function (Cipollo and Trimble, 2002). Results In many of the studies from this laboratory, highly glycosylated S. cerevisiae external invertase has been used as a source of N-glycans for oligosaccharide structural studies. Because the enzyme represents an end product of the secretory pathway, it provides a useful probe for analyzing the effect of N-glycosylation pathway mutations on the downstream processing and function of glycoprotein glycans. Initial studies 750 to define alg12 N-glycan structures using external invertase as a source revealed that in contrast to wild-type or alg3-1 cells, where invertase derepression induces a high level of stable activity, the activity in alg12 cells was unexpectedly low and not stable over time. In alg12 cell extracts, the total invertase activity initially present declined rapidly, indicating that both the internal and external forms were being proteolytically degraded (Cipollo and Trimble, 2002). Characterization of alg12 invertase By monitoring PrA activity after derepression for invertase and adding pepstatin A to 3 µm when protease activity began to rise (Cipollo and Trimble, 2002), alg12 yeast transformed with the prb58 plasmid (Verostek et al., 1991) overproduced SUC2 invertase at a level exceeding 700 IU/g (wet weight). This yielded a high specific activity external invertase (68 mg, 3670 U/mg protein) with an overall recovery of 60% from 635 g of cells. A western blot of wild-type, alg12 -, and alg9 -secreted invertases revealed wild-type external invertase to migrate as a diffuse band with an average apparent mass of ~120 kda, whereas external invertases from alg12 and alg9 were progressively more heterogeneous in mass with average weights of 110 kda and 95 kda, respectively (data not shown).

3 N-glycan processing in alg12 S. cerevisiae Bio-Gel P-4 size-exclusion chromatography of the peptide-nglycosidase F (PNGase F) released glycans from the alg12 invertase preparation provided four major pools, labeled A through D in Figure 1, which eluted on the calibrated column in volumes consistent with Hex 7 GlcNAc 2 (fractions ), Hex 8 GlcNAc 2 (fractions ), Hex 9.5 GlcNAc 2 (fractions ), and Hex 11 GlcNAc 2 (fractions ), respectively. Each pool was rechromatographed, the central 85% of the resulting peaks were repooled (data not shown), and the sizes of the glycans in each were confirmed by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) (Figure 2). Pool A was solely Hex 7 GlcNAc 2. Pool B revealed glycan ions for Hex 8 GlcNAc 2 and Hex 9 GlcNAc 2. Although MS analysis is not generally considered quantitative, signal intensities from adjacent structures in a homologous structural series have been shown to be proportional (Cipollo and Trimble, 2000). On this basis, Hex 8 GlcNAc 2 was 80% of pool B and Hex 9 GlcNAc 2 was the remaining 20%. MS analysis of pool C revealed the presence of 60% Hex 9 GlcNAc 2 and 40% Hex 10 GlcNAc 2, and pool D was approximately 30% Hex 10 GlcNAc 2 and 70% Hex 11 GlcNAc 2. High-pH anion-exchange chromatography (HPAEC) analysis To estimate the number of branch isomers present in the four oligosaccharide pools, each was analyzed by Dionex HPAEC using a CarboPak PA-100 column. Pool A gave a major peak (Figure 3A) that coeluted with the smallest alg12 glycan released from OSL (data not shown), consistent with the hypothesis that the alg12 core isomer (Scheme 1C) is unmodified by further processing after transfer to protein and transport through the secretory pathway. Two minor peaks, whose structures will be elucidated, were detected. Pool B yielded four peaks (Figure 3B) corresponding to the presence of a minimum of four branch isomers, with the most abundant glycan peak representing ~49% of the profile area. The remaining three minor peaks contained approximately 22%, 22%, and 7% of the total profile area, respectively. In pool C (Figure 3C) two major and two minor peaks were evident. The relative integrated peak intensities gave an estimated distribution of 37%, 35%, 19%, and 9%. The peaks representing 37% and 35% (see Figure 3) of the integrated peak area eluted at 15 min and 18.5 min, respectively, and appeared to contain Fig. 1. Bio-Gel P-4 (extra-fine mesh) chromatography of PNGase F released alg12 invertase oligosaccharides. The glycans separated into large Vo oligosaccharides (open triangles) and peaks in the size range of Hex 7 11GlcNAc 2 (closed triangles). Glc 3 [ 3 H]Man 9 GlcNAc 2 (circles) was included as an internal marker. Samples were assayed for phenol-sulfuric acid color and liquid scintillation counting. Fractions were pooled as indicated. Fig. 2. MALDI-TOF MS of S. cerevisiae alg12 Hex 7 11 GlcNAc 2 pools A D. The masses of the sodium adducts of the size isomers were: Hex 7 GlcNAc 2, 1582 Da; Hex 8 GlcNAc 2, 1744 Da; Hex 9 GlcNAc 2, 1906 Da; Hex 10 GlcNAc 2, 2068 Da; and Hex 11 GlcNAc 2, 2230 Da. more than one component. Pool D (Figure 3D) yielded five glycan peaks, with the main species representing 60% of the total integrated area. The additional peaks accounted for approximately 16%, 10%, 10%, and 4% of the total peak area present in the HPAEC profile. Nuclear magnetic resonance (NMR) analysis of alg12 core oligosaccharides To determine the glycan structures present in Bio-Gel P-4 pools A D, 1D 1 H NMR spectra (not shown) were collected as in previous studies (Cipollo et al., 2001; Verostek et al., 1993a,b), and anomeric and selected C2-H protons were integrated for each at 300 K. For accurate integration of residue 3, 1D NMR spectra were collected at 318 K, which moves the HOD signal upfield to ppm. Integration of proton intensities for established reporter groups present in expansions of the spectra are summarized in Table I. All structures assigned for alg12 glycans in the current work had as their core GlcNAc residues 1 and 2 and Man residues 3 5, 7, 8, and11 (Scheme 1B). However, 7 s resonance intensity can appear at either 5.11 ppm in its unsubstituted form or at 5.41 ppm if it is 2-O-substituted with Man 10 (Byrd et al., 1982; Haeuw et al., 751

4 J.F. Cipollo and R.B. Trimble from4.13to4.15ppm(winniket al., 1982; Hard et al., 1991), whereas its C3-H would remain centered at ~3.91 ppm (Ziegler et al., 1999). Fig. 3. Analytical HPAEC of individual S. cerevisiae alg12 Hex 7 11 GlcNAc 2 pools. Approximately 3 nmol of total glycan were loaded on a mm PA-100 column and chromatographed as described in Materials and methods. 1991; Cipollo and Trimble, 2000). The addition of residue 10 to residue 7 is catalyzed by Alg9p (Burda et al., 1996; Cipollo and Trimble, 2000) and its hydrolysis is catalyzed in the ER by Mns1p in wild-type cells (Ziegler and Trimble, 1991). Fractional molar proton intensities in pools A D (Table I) were assigned as additions to the core structure (Scheme 1B), resulting in a homologous series of structurally related compounds (summarized in Scheme 2). For reference, the alg9 Man 6 GlcNAc 2 core glycan, which contains all core structure residues, is included in Table I. The isomer identifications in Scheme 2 relate the hexose number present and the order in which fractional proton intensities were used to deduce the structures; for example, isomer 8a denotes the first Hex 8 GlcNAc 2 configuration assigned in pool B. As will be documented in subsequent sections, the number and amount of each isomer assigned within each pool agreed closely with the number and area of HPAEC peaks present (Figure 3) and the size proportions estimated by MALDI-TOF MS (Figure 2). Some assignments required double quantum filtered correlation spectroscopy (DQF COSY) experiments for validation, which are presented in Figures 4 and 5. The C2-H resonance intensities at 4.22 ppm and 4.14 ppm were key signatures for the structural assignment of some pool components. Proton intensity at 4.22 ppm arises from 3-O-substituted α1,2-man residues 10, 11, and13; 3-O- substituted α1,3-man residues 14, 15, and16; and residue 3 in the absence of a through-space effect occurring when residue 5 is 6-O-substituted by 12 (Scheme 1D; Haeuw et al., 1991; Trimble and Atkinson, 1992; Verostek et al., 1993b; Cipollo and Trimble, 2000). Resonance intensity at 4.14 ppm arises from residue 3 s C2-H being shifted upfield by a through-space effect caused when 12 is present on 5 or from 4 s C2-H, when 3-O-substituted by residue 7 (Scheme 1; Trimble and Atkinson, 1992; Ziegler et al., 1999). Residue 7 was present in all isomers, and the sum of its anomeric proton intensities at 5.11 and 5.41 ppm equaled 1 mol in all pools, which means that all of residue 4 was 3-Osubstituted by 7, providing 1 mol of C2-H intensity at 4.14 ppm. By subtraction, any intensity at 4.14 ppm in excess of 1 mol must be due to 3 s C2-H when residue 12 substitutes 5 (see Scheme 1D). Importantly, the resonance signal for upper-arm residue 6 or its 2-O-substituting Man residue 9 was not detected on any ofthenmrspectra.thepresenceofresidue6 would shift 4 s C1-H from 4.89 to 4.87 ppm, accompanied by a shift in its C2-H 752 Pool A: Hex 7 GlcNAc 2. Integration of proton resonance intensity for pool A between 4.70 and 5.45 ppm provided 7.00 mol of hexose protons (Table I). Compared to the core Man 6 reference isomer described (Scheme 1B and Table I), the alg12 pool A provided 1 mol of added proton resonance intensity, which was divided among peaks found at 4.91, 5.04, and 5.14 ppm. The 0.87 mol of proton intensity at 5.41 ppm is assigned to the core 2-O-substituted residue 7 (Scheme 1C), which is confirmed by the 2D DQF COSY cross-peak seen at 5.41 (C1-H)/4.08 (C2-H) ppm (Figure 4A; Byrd et al., 1982; Haeuw et al., 1991). The 0.87 mol of signal at 5.04 ppm is from residue 10, which 2-O-substitutes residue 7; its presence is confirmed by the 2D DQF COSY J 1,2 cross-peak at 5.04 (C1-H)/4.08 (C2-H) ppm (Figure 4A; Trimble and Atkinson, 1992). The proton resonance allocations assign the major pool A component as the predicted Man 7 GlcNAc 2 isomer 7a (Scheme 2), which is identical to the alg9 Man 6 GlcNAc 2 precursor (Cipollo and Trimble, 2000) with α1,2-man residue 10 substituting residue 7. The 0.13 mol of proton resonance at 5.11 ppm is assigned to unsubstituted α1,3-linked residue 7, which means that 13% of pool A is isomers without α1,2-man 10. The remaining 0.13 mol of proton resonance consistent with Man 7 GlcNAc was seen at 4.93 ppm (0.03 mol) and 5.14 ppm (0.10 mol) for α1,6-man residue 12 and α1,3-man 14, respectively (Trimble and Atkinson, 1992), allowing assignment of isomers 7b (3%) and 7c (10%) (Scheme 2). All of the resonance intensity centered at 4.22 ppm (1.07 mol; Table I) is wholly accounted for by residue 3 of isomer 7a (0.87 mol) and residues 3 (0.10 mol) and 14 (0.10mol)ofglycan7c(see Schemes 1 and 2). These assignments are consistent with the number of peaks and integrated area seen by HPAEC analysis and the size isomer distribution estimated by MALDI-TOF MS (Figure 2). Pool B: Hex 8 9 GlcNAc 2. The anomeric proton intensity in pool B was 8.27 mol (Table I), indicating that 73% of the isomers present were Hex 8 GlcNAc 2, and 27% were Hex 9 GlcNAc 2,in good agreement with the MALDI-TOF MS distribution estimated in Figure 4B to be 80% Hex 8 GlcNAc 2 and 20% Hex 9 GlcNAc 2. C1-H proton intensity in excess of the core Man 6 reference isomer (Scheme 1B and Table I) was present at chemical shifts at 4.89 ppm (0.09 mol), 5.04 ppm (1.18 mol), ppm (0.91 mol), and 5.25 ppm (0.10 mol). Core residue 7 was found distributed as 0.85 mol at 5.41 ppm, and the remaining 0.15 mol at 5.11 ppm (Table I). Thus, as assigned and as documented by the 2D DQF COSY cross-peak at 5.41 (C1- H)/4.08 (C2-H) ppm (Figure 4B), 85% of the isomers present have α1,2-man 10 on Man 7. At 4.14 ppm the total proton intensity was 1.42 mol (Table I), which included 1.00 mol from residue 4 and 0.42 mol from residue 3 due to the through-space effect on 3 caused by 5 s 6-O-substitution with residue 12. Thus, 42% ofpool-b isomers contain residue 12. The 0.09 mol of resonance intensity seen at 4.92 ppm (Table I) is from unsubstituted 12. The assignment was confirmed by a low cut in the C1-H/C2-H region of the DQF COSY spectrum, which revealed a cross-peak centered at 4.92 (C1-H)/3.91(C2-H) ppm (not seen in Figure 4B; Trimble

5 N-glycan processing in alg12 S. cerevisiae Table I. C1-H and C2-H intensity integrations for alg12 external invertase glycan pools A D Nominal chemical Relative molar proton intensity in Residue no. Linkage a shift (δ, ppm) Alg Man 6 Pool A Pool B Pool C Pool D C1-H 7 Man Man Man G 1 Glc Tr 1α GlcNAcα β4 7 Man /15/16/ Man /15/16 Man /11/13 Man /11/13 Man Man Manβ c 1.00 c 1.00 c 1.00 c 3,6 1β GlcNAcβ β4 2 GlcNAcβ β4 C2-H 3 Manβ , Man /15/16/18 Man /11/13 Man Total mol hexose C1-H a All linkages are alpha unless otherwise indicated. b Integrated intensities are taken from the alg9 Man 6 GlcNAc 2 1 H NMR data in Cipollo and Trimble (2000). c Core residue 3 was integrated at 318K. and Atkinson, 1992; Cipollo and Trimble, 2000). Subtracting the 0.09 mol of unsubstituted 12 from the 0.42 mol total for 12 leaves 0.33 mol of 12 2-O-substituted by 13. These allocations necessitate that in addition to the core residues 1 5, 7, 8, and 11, the following can be made as partial structural assignments: 9% have terminally linked 12, 33% have 12 and its 753

6 J.F. Cipollo and R.B. Trimble Scheme 2. Interrelationship of alg12 Hex 7 11 GlcNAc 2 species deduced in this study. The identifiers for structures are those used in the text. The ER and Golgi regions of the secretory pathway in which the reactions occur are indicated at the top. Glycan families of interest are: (A) N-glycans that retain a single Glc; (B) mature wild-type core-filled isomers or substrates for elongation to mature core-filled isomers; (C) minimally processed core-type glycans that escape Och1p activity; (D) alg9-type core-filled species. The asterisk appears above the glucosylated structures deduced from in vivo [2-3 H]Man-labeling studies (see text). The Alg3p substrate Man 5 GlcNAc 2 and Alg9p substrate Man 6 GlcNAc 2 from which all structures in this study are derived are indicated by and, respectively. Structures 9x and 10x were not seen in the glycan pools but are expected intermediates leading to the synthesis of isomer 11b. 2-O-substituent residue 13, and 58% are minimally processed core type with no added 12 or 13. At 5.04 ppm the 1.18 mol of proton intensity is divided between terminal and 3-O-substituted α1,2-man, whose presences are verified by the 2D DQF COSY cross-peaks seen at 5.04 (C1-H)/4.06(C2-H) ppm and 5.03(C1-H)/4.22(C2-H) ppm, respectively (Figure 4B; Haeuw et al., 1991; Verostek et al., 1993b; Cipollo and Trimble, 2000). Of this resonance intensity, 0.85 mol was assigned to residue 10, leaving 0.33 mol of this signature to assign. For the Hex 8 9 GlcNAc 2 -sized compounds studied here, added proton resonance intensity, above that seen for residue 10, can only come from residue 13 (Scheme 1D), which 2-O-substitutes residue 12 (Trimble et al., 1991; Trimble and Atkinson, 1992; Cipollo and Trimble, 2000). This indicates that 33% of pool isomers have residue 13, coinciding with the amount assigned using the C2-H intensities at 4.14 ppm. 754 Between 5.10 and 5.14 ppm, 1.06 mol of resonance intensity in excess of that attributed to the core Man 6 GlcNAc 2 was integrated. Of that 1.06 mol, 0.15 mol of intensity was present at 5.11 ppm. Subtracting the 0.15 mol of unsubstituted 7 s intensity at 5.11 ppm (see previous discussion) from the 1.06 mol of signal in the ppm region leaves 0.91 mol to assign. A 2D DQF COSY cross-peak seen at 5.14 (C1-H)/4.03(C2-H) ppm (Figure 4B) is characteristic of α1,6-man 12 when 2-Osubstituted by residue 13 (Verostek et al., 1993b; Cipollo and Trimble, 2000) and accounts for 0.33 mol of the remaining 0.91 mol of intensity, leaving 0.58 mol to assign. The final 2D DQF COSY cross-peak detected from anomeric protons between 5.10 and 5.15 ppm was at 5.14(C1-H)/4.06(C2-H) ppm (Figure 4B), due to terminally linked α1,3-man, and accounts for the remaining 0.58 mol of proton intensity. In the compounds with the Man 7 GlcNAc 2 core studied here, this

7 N-glycan processing in alg12 S. cerevisiae Fig. 4. 2D DQF COSY 1 H NMR spectra C1H C2H cross-peak regions of pools A D. Panels A D represent the respective pools. The residue and linkage type of each peak are indicated. Linkage key: 1α,GlcNAcα;1β, GlcNAcβ;2t,Manα1,2-; 2i 3,Manα1-3Manα1,2-; 3t, Manα1,3-; 3i 3,Manα1,3Manα1,3-; 6t, Manα1,6-; 6i 2,Manα1,2Manα1,6-; β4i 3,6,Manα1,6(Manα1,3)Manβ1,4-; β4i 3i 6,6,Manα1,6(Manα1,6Manα1,3)Manβ1,4-; the underscore designates the residue described by the indicated symbol. resonance can arise from mannoses 14, 15, 16, and 18 (Scheme 1D; Cipollo and Trimble, 2000). At 5.25 ppm 0.10 mol of resonance intensity was integrated. The 2D DQF COSY spectrum reveals a cross-peak at (C1-H)/3.56(C2-H) ppm, confirming the presence of residue G 1 (Trimble and Atkinson, 1986; Verostek et al., 1993b; Cipollo and Trimble, 2000). This indicates that 10% of the pool s isomers retained the innermost α1,3-linked Glc residue on the lower-arm Man residue 11 (Scheme 1A). At 4.22 ppm 1.26 mol of C2-H intensity was integrated. Of this, 0.58 mol is from 3 in the absence of 12 as assigned. Another 0.10 mol is from residue G 1, leaving 0.58 mol attributed to the fractional 3-O-substitution by residues 14, 15, and/or 16 of residues 10, 11, and/or 13 (see Scheme 1D), which is in agreement with the assignment of 0.58 mol of α1,3-man residues at 5.14 ppm derived earlier. Of the 0.58 mol of Man 6 GlcNAc 2 core in pool B that does not have residue 12 (Scheme 1B), 0.48 mol of 10 and an equal amount of the terminal α1,3-man residue 14/15, are assigned, defining 48% of the pool as isomer 8a (Scheme 2). The remaining 0.10 mol of 3-O-substituted Man, 0.10 mol of 10, and 0.10 mol of G 1 are assigned to isomer 8b as 10% of the pool B isomers. This accounts for all of the 3-O-substituted Man C2-H at 4.22 ppm and leaves 0.27 mol of the 0.85 mol of 10 to assign. For the 33% of the pool components 755

8 J.F. Cipollo and R.B. Trimble Fig. 5. 2D DQF COSY 1 H NMR spectra C2H-C3H cross-peak regions of pools A D. The residue and linkage type of each peak are indicated as in the legend to Figure 4. The boxed inset is a lower cut of this region of the spectrum revealing residue 3 unsubstituted by 12. with residues 13 and 12 as assigned, 15% are without residue 10 defining glycan 8c. The remaining 18% of the isomers with residues 12 and 13 have residue 10, assigning isomer 9a (Scheme 2), which leaves 0.09 mol of 10 to assign. Isomer 8d with terminally linked Man 12 represents 9% of pool B and consumes 0.09 mol of remaining residue 10 (Scheme 2). The HPAEC profile of the pool B gave a distribution of 49%, 22%, 22%, and 7% of total area with a pronounced shoulder on the peak eluting at 16 min (Figure 3B). This is consistent with the presence of two isomers having similar elution characteristics. Thus the 1 H NMR assignments are in close agreement with the 756 isomer distribution predicted by HPAEC analysis. Pool C: Hex 9 10 GlcNAc 2. The total Man and Glc anomeric proton intensity of pool C was 9.35 mol, which corresponds to 65% Hex 9 GlcNAc 2 and 35% Hex 10 GlcNAc 2 (Table I). These values are in close agreement with the MS profile, which estimated 60% Hex 9 GlcNAc 2 and 40% Hex 10 GlcNAc 2 (Figure 2C). In pool C the proton intensity of the Man 6 core (Scheme 1B) was 3.35 mol, which included 0.17 mol between 4.89 and 4.92 ppm for unsubstituted α1,6-man residue 12 (see Scheme 1D); 1.35 mol at 5.04 ppm for 2-O-linked Man residues 10 and

9 N-glycan processing in alg12 S. cerevisiae Fig. 6. Analytical HPAEC of N-linked oligosaccharides from alg12 glycoproteins pulse/chase-labeled in vivo with [2-3 H]Man. Mid-log cells were labeled for 2 min with [2-3 H]Manintheabsence(A C) orpresence(d F)of 5 mm CST and chased with excess unlabeled Man. Aliquots from each reaction were terminated by addition of CHCl 3 /CH 3 OH (2:1) at 0 (A, D), 1(B, E), or 10 (C, F) min of chase. All pellets were solubilized; N-glycans werereleased with endo H and then characterized on a PA-100 column previously calibrated with authentic oligosaccharide standards from this and an earlier study (Verostek et al., 1993b). Solid lines are protein-released glycans. In A and D broken lines are an alg12 Man 7 GlcNAc standard releasedfromoslanddigestedwithendoh.additionaldetailsarein Materials and methods. 13; 1.79 mol at 5.14 ppm for α1,3-man residues 14, 15, 16 and/ or 18; andα1,6-man 12 when 2-O-substituted with residue 13. The C2-H proton intensity at 4.22 ppm was 1.58 mol, and the intensity found at 4.14 ppm equaled 1.71 mol (Scheme 1B and Table I). The anomeric proton intensity at 5.41 ppm for 2-O-substituted 7 was 0.81 mol, which was verified by the strong 2D DQF COSY cross-peak at 5.41(C1-H)/4.10(C2-H) ppm (Figure 4C), indicating that 80% of pool C isomers have residue 10. The remaining 0.19 mol of residue 7 s anomeric proton was present at 5.11 ppm, characteristic of its unsubstituted form. At 4.14 ppm 1.71 mol of C2-H proton intensity was found. As described for previously assigned isomers, this signal intensity is from residues 3 (when 12 is present) and 4. Because 4 contributes 1.00 mol of intensity, 3 contributes the remaining 0.71 mol, indicating that 71% of the pool isomers contain residue 12. The 0.17 mol of added signal seen at 4.92 ppm means that 17% of the pool isomers had an unsubstituted residue 12, verified by a low cut of the 2D DQF COSY spectrum, which revealed the 4.92 (C1-H)/3.98(C2-H) ppm cross-peak (seen in Figure 4C; Verostek et al., 1993b; Cipollo and Trimble, 2000). By difference ( mol), 0.54 mol of 12 is 2-O-substituted with 13, whose cross-peak is seen at 5.14 (C1-H)/4.02(C2-H) ppm in Figure 4C. This allows the following partial structural assignments: 54% of the pool glycans contain 12 and 13, 17% contain terminally linked 12, and 29% are devoid of 12 and 13 (Table I and Scheme 1). The 1.35 mol anomeric proton intensity seen at 5.04 ppm (Table I) is divided between terminal and 3-O-substituted α1,2-man residues 10 and 13. The 2D DQF COSY J 1,2 crosspeaks at 5.04 (C1-H)/4.07(C2-H) ppm and 5.03(C1-H)/ 4.22(C2-H) ppm (Figure 4C) verify the presence of both unsubstituted and 3-O-substituted α1,2-man residues, respectively (Trimble and Atkinson, 1992; Verostek et al., 1993a,b; Cipollo and Trimble, 2000). Because 0.81 mol is present as α1,2-man 10, which is equal to the intensity of 2-O-substituted 7 at 5.41 ppm in Table I, by subtraction 0.54 mol of proton intensity is from terminal α1,2-man 13. These assignments confirm the presence of 13 and 12 on 54% of the pool C isomers as calculated in the previous paragraph. As observed from pool C s 2D DQF COSY spectrum (Figures 4C and 5C), 1.79 mol of proton intensity above the Man 6 core was present at 5.14 ppm and was distributed between two residue linkage types. The defining cross-peaks were found at 5.14 (C1-H)/4.02 (C2-H) ppm and 4.02 (C2-H)/ 3.92 (C3-H) ppm for the 2-O-substituted α1,6-linked residue 12 (Trimble and Atkinson, 1992) and 5.14 (C1-H)/4.07 (C2-H) ppm and 4.07 (C2-H)/3.88 (C3-H) ppm for terminal α1,3-man. Subtracting 0.54 mol of 13 from the 1.79 mol of intensity at 5.14 ppm defines 1.25 mol of α1,3-man. At 5.25 ppm, 0.04 mol of proton intensity was detected for G 1 (Table I). Although not apparent in Figure 4C, this assignment is confirmed by a near baseline cut in pool C s 2D DQF COSY spectrum, which revealed the defining cross-peak at 5.25 (C1-H)/3.54 (C2-H) ppm (Trimble and Atkinson, 1986; Verostek et al., 1993b). Of the 1.58 mol of C2-H intensity seen at 4.22 ppm, 0.29 mol was from residue 3 in structures lacking residue 12, leaving 1.25 mol arising from 3-O-substituted residues 14, 15, and16, and 0.04 mol from residue 11 3-O-substituted by G 1.Toa portion of pool C isomers with residues 12 and 13 (54%), we assign 0.35 mol of 3-O-substituted Man and an equal amount of 5.04 ppm resonance from the 0.81 mol of residue 10, defining 35% of the pool as the major isomer 10a. This leaves 0.90 mol of 3-O-substituted Man and 0.46 mol of Man 10 to assign. To the remaining 19% of pool C s isomers having residues 12 and 13 was assigned 0.19 mol of terminal α1,3-man, defining pool B isomer 9b, and leaving 0.71 mol of resonance at 4.22 ppm, equal to the remaining α1,3-man to assign. Note that isomer 9b accounts for all of α1,3-man 7 as a terminal residue, whose resonance was detected at 5.11 ppm as defined earlier. To 17% of the pool isomer with unsubstituted residue 12 (the 0.17 mol signal at 4.92 ppm in Table I) was assigned 0.17 mol of both α1,3-man and residue 10 to give isomer 9c. This leaves 0.54 and 0.29 mol, respectively, of each residue to assign. To the 29% Man 6 core structure (Scheme 1B) that did not have residues 12 or 13 described earlier, we assigned 0.25 mol of the remaining 10 and 0.50 mol of α1,3-man, defining isomer 9d. This leaves 0.04 mol each of G 1, α1,3-man, residue 10, andtheman 6 glycan, which together define isomer 9e (4%), thus completing the assignment of the pool s components. All structures can be seen in Scheme 2. The number and distribution of isomers in the pool as derived by proton intensity allocation (Table I) are in good agreement with the isomer number and distribution predicted by HPAEC and estimated Hex 9 /Hex 10 MS intensities (60/40). Pool D: Hex GlcNAc 2. pool. The Man and Glc anomeric proton intensity above the Man 6 core in pool D (Scheme 1 and Table I) was 4.60 mol, giving a total of mol of proton intensity. This indicates that the pool contains 60% Hex 11 GlcNAc 2 and 40% Hex 10 GlcNAc 2, which is in close agreement with the MALDI-TOF MS prediction of 70% Hex 11 GlcNAc 2 and 30% Hex 10 GlcNAc 2 (Figure 2D). The 757

10 J.F. Cipollo and R.B. Trimble increased C1-H resonance intensity above that provided by the core Man 6 GlcNAc 2 was observed at 5.25 ppm (trace), 5.14 ppm (2.70 mol), 5.04 ppm (1.75 mol), and 4.92 ppm (0.15 mol). The 0.90 mol of signal at 5.41 ppm is from core residue 7 2-O-substituted by 10, which is verified by the characteristic 2D DQF COSY cross-peak at 5.41 (C1-H)/4.10 (C2-H) ppm (Figure 4D). Subtracting 0.90 mol of residue 10 from the 1.75 mol of signal at 5.04 ppm for α1,2-linked Man leaves 0.85 mol, which is assigned to α1,2-man 13 substituting 12. This assignment is supported by the strong 2D DQF COSY cross-peak at 5.04 (C1-H)/4.07 (C2-H) ppm (Figure 4D), the signature of terminal α1,2-man. Another cross-peak at 5.03 (C1-H)/4.22 (C2-H) ppm was observed for 3-O-substituted α1,2-man (Figure 4D), indicating that a portion of the pool s isomers have α1,3-man residues 14, 15, and/or16. Thus, 90% of pool D s glycans have residue 10 and 85% have α1,2-man 13, which 2-O-substitutes Man 12. At 4.91 ppm, 0.15 mol of resonance intensity in excess of the Man 6 core residue 4 was integrated. The assignment is supported by the presence of a 2D DQF COSY cross-peak of low intensity at 4.91 (C1-H)/4.02 (C2-H) ppm (not apparent in Figure 4D). Thus all of pool D s isomers have residue 12;15% unsubstituted and 85% 2-O-substituted with 13 (Table I). The 2.80 mol of resonance at ppm that provided by the core Man 6 structure includes 0.10 mol from core residue 7 not substituted by 10. This amount is derived by subtracting 0.90 mol of 2-O-substituted 7 at 5.41 ppm from unity, in agreement with integration of ~0.10 mol of resonance intensity at 5.11 ppm in the 1D NMR spectrum (Table I). An additional 0.85 mol of intensity in this chemical shift region of the spectrum is from 2-O-substituted 12 (described earlier). The remaining intensity at ppm (1.85 mol) is assigned to α1,3-linked residues 14, 15, 16,and18 (Scheme 1D). A strong cross-peak in the 2D DQF COSY spectrum of pool D at 5.14 (C1-H)/4.06 (C2-H) ppm (Figure 4D) confirms the presence of these terminal α1,3-linked residues. At 4.22 (C2-H)/4.01 (C3-H) ppm (Figure 5D), a low-intensity J 2,3 cross-peak is present, indicating that a small amount of 14, 15, and/or 16 is 3-Osubstituted with residue 18 (Verostek et al., 1993b; Cipollo and Trimble, 2000). Note in the J 2,3 region of the 2D DQF COSY spectrum the absence of a cross-peak for residue 3 at 4.22 (C2-H)/4.01 (C3-H) ppm (compare Figures 5A D), thus confirming that all of pool D sisomershave residue 12. With3 s C2-H shifted upfield, all of the assigned 4.22 ppm signal (1.85 mol) must be from 3-Osubstituted α1,2-man and α1,3-man residues. The 2.00 mol C2-H resonance intensity at 4.14 ppm is for the C2-Hs of 3 and 4, as expected (Table I). Distribution of the integrated protons and a Hex GlcNAc 2 size constraint allow assignment of the two major isomers in pool D as 11a (60%) and 10a (15%) (Scheme 2). These structures account for 1.35 mol of resonance intensity at 4.22 ppm, leaving 0.50 mol to assign. Isomers 10b (15%) and 10c (10%) account for the remaining 0.5 mol of 4.22 ppm resonance intensity (see Scheme 2 for isomers). In addition, a trace amount of G 1 s anomeric proton was detected at 5.25 ppm in pool D s 1D NMR spectrum (Table I), which leads to the assignment of a trace of isomer 11b. These assignments are in good agreement with the pool s HPAEC profile, which gave a predicted isomer distribution of 60%, 16%, 10%, 10%, and 4% (Figure 3). 758 In vivo glucosylation of alg12 OSL NMR-derived structures of secreted invertase glycans in alg12 yeast indicate that ~4% of Hex 7 11 GlcNAc 2 isomers retained residue G 1 (Scheme 2). To ascertain whether G 1 was a product of compromised OSL glucosylation and impaired Glc trimming on nascent glycoproteins, as seen in alg3-1 yeast (Verostek et al., 1991), or a remnant of full glucosylation and processing as seen in alg9 cells (Cipollo and Trimble, 2000), alg12 cells were pulse-labeled for 2 min with [2-3 H]Man in the absence (Figures 6A C) or presence (Figures 6D F) of the glucosidase inhibitor castanospermine (CST) and the label chased with unlabeled Man for 0, 1, or 10 min. The labeled glycans were released from glycoprotein pellets by endoglycosidase H (endo H) and analyzed by HPAEC as described in Materials and methods. The additional trace in Figure 6A and D (dashed line) is overlayed from a separate run of a [ 3 H]Man 7 GlcNAc standard isolated from alg12 OSL and treated with endo H to remove one GlcNAc. After a 2-min [2-3 H]Man pulse in the absence of CST, a complex peak of glycans was present in the HPAEC profile at min (Figure 6A), consistent with a composition of Glc 2 Man 7 GlcNAc as well as Man 8 10 GlcNAc elongated from the trimmed Man 7 GlcNAc precursor. Note, however, in the present of CST essentially all of the glycan label was trapped by the glucosidase inhibitor in Glc 3 Man 7 GlcNAc (Figure 6D) eluting at 26 min. This 26-min peak generated a Hex 7 GlcNAcsized product on jackbean α-mannosidase digestion (data not shown), consistent with removal of Man residues 10, 7, and4 from the fully glucosylated alg12 Glc 3 Man 7 GlcNAc core (Scheme 1A). The large run-through radioactivity is [2-3 H]Man, which could not be completely removed from the cell pellets by washing prior to making extracts. Comparison of the glycan profiles in Figures 6A and 6D implies that in alg12 essentially all of the OSL is fully glucosylated, and, following transfer to protein, the glucoses are rapidly and efficiently removed. This occurs so quickly, in fact, that during the 2-min pulse in the absence of CST (Figure 6A), one to three Glc residues were removed from nearly all the labeled glycans and a portion coelute with the [ 3 H]Man 7 GlcNAc external standard ( ). In the presence of CST, some of the Glc 3 Man 7 GlcNAc has already been elongated to larger species eluting by HPAEC at min (Figure 6D). After only 1 min of chase with unlabeled Man almost all of glucosylated glycans were either deglucosylated (Figure 6B) or elongated to larger forms (Figure 6E). Since the t 1/2 of protein secretion in yeast is about 5 min (Franzusoff, 1992), and no labeled ER forms of glycoproteins are seen after a 10-min chase (Franzusoff and Schekman, 1989), the glycans found after the 10-min chase in Figures 6C and F are from glycoproteins trafficked to the Golgi and beyond. These constitute elongated species (the Vo glycans in Figure 1), which elute on column regeneration (Cipollo and Trimble, 2000) and shorter glycans eluting from 7 to 40± min, which include the species present in Bio-Gel P-4 pools A D (Figures 1 and 3). After2minoflabelinginboththepresenceandabsenceof CST, a peak was seen in the HPAEC profile at 10 min (Figure 6A and D). Because no Glc trimming intermediates were seen between 10 and 26 min in the elution profile immediately after the 2-min pulse in the presence of CST (Figure 6D), it is likely this peak represents a small amount of Glc 1 Man 7 GlcNAc 2 that

11 N-glycan processing in alg12 S. cerevisiae was transferred directly to protein in the alg12 background under conditions in which Glc 3 Man 7 GlcNAc 2 -PP-Dol availability may have been limiting. Bio-Gel P-4 analysis of [ 3 H]Manlabeled glycans from the OSL fraction from cells labeled during the pulse-chase experiment revealed that the relative amount of Glc 3 Man 7 GlcNAc 2 -PP-Dol was low (Figure 6A), whereas the amount of glycan eluting at 10 min was significant, consistent with Glc 1 Man 7 GlcNAc 2 -PP-Dol (Figure 6A C). However, the amount of labeled glycan eluting at 10 min in the HPAEC profiles was insufficient to structurally confirm this peak as Glc 1 Man 7 GlcNAc. Nevertheless, it is worth noting that Glc 1 Man 7 GlcNAc 2 and Glc 1 Man 8 GlcNAc 2 were among invertase glycans present in pools B and C (Scheme 2) at levels that could easily account for the [2-3 H]Man-labeled glycan peak(s) eluting at 8 11 min in Figure 6. Discussion In this report we have established that the core alg12 Man 7 GlcNAc 2 released from external invertase is Man α1,2manα1,2 Manα1,3 (Manα1,2 Manα1,3 Manα1,6)-Manβ1,4GlcNAcβ1,4GlcNAcα/β (Scheme 1C), confirming the structure deduced for the Man 7 GlcNAc 2 released from alg12 OSL characterized by high-performance liquid chromatography (HPLC) and α1,2-mannosidase digestion by Burda et al. (1999). This means that the Man 7 GlcNAc 2 that accumulates in alg12 is the direct product of the Alg9p α1,2-mannoslytransferase. The structures of Golgi-processed N-glycans from external invertase assigned in the present study are summarized in Scheme 2 and provide insight regarding the role of the ALG9 step in downstream ER and Golgi N-glycan processing events. Overexpression of Alg12p in the alg9 background forms, in addition to the alg9 Man 6 OSL (Scheme 1B), a novel Man 7 OSL, Manα1,2Manα1,2Manα1,3 (Man1,6(Manα1,3)Manα1,6) Manα1,4GlcNAcα1,4GlcNAc-PP-Dol, consistent with the ALG12 locus encoding the Man-P-Dol:Man 7 GlcNAc 2 -PP-Dol α1,6-mannosyltransferase that adds the upper-arm α1,6-linked Man residue 6 (Scheme 1A) to OSL (Burda et al., 1999). Apparently, overexpression of Alg12p drives the addition of some α1,6-linked Man residue 6 in the absence of the centralarm α1,2-linked Man 10 (Scheme 1A), whose addition by Alg9p normally precedes it (Hubbard and Robbins, 1980). What is particularly interesting about this observation is that the absence of α1,2-man 9 on this Man 7 glycan, characterized by HPLC size and α1,2-mannosidase sensitivity, suggests that Alg9p adds both residues 10 and 9 to wild-type OSL. Noteworthy in this regard, a candidate enzyme to add the last α1,2-manto OSL (residue 9, Scheme 1A) has not been identified by genetic or homology searching methods. In this study, addition of residue 6 in alg12 yeast with normal levels of Alg9p could not be detected. The J 2,3 and J 3,4 coupling constants of mannose residues in polysaccharides such as those studied here are ~3.5 Hz and ~10.0 Hz, respectively, giving rise to strong C2-H/C3-H 2D DQF COSY crosspeaks, allowing detection of trace amounts of such residues. The absence of any resonance for residue 4 at 4.15 (C2-H)/3.91 (C3-H) ppm (Winnik et al., 1982) or that for 2-O-substituted residue 6 at 4.03 (C2-H)/3.96 (C3-H) ppm (Trimble and Atkinson, 1992) verifies a paucity of 6. This means that Alg12p is required for addition of residue 6 in vivo, which suggests that under normal growth conditions yeast carefully regulate the level of the OSL mannosyltransferases to ensure ordered assembly of Glc 3 Man 9 GlcNAc 2 -PP-Dol (Burda et al., 1999; Jakob et al., 1998). Glucosylation of Man 5 GlcNAc 2 -PP-Dol in the alg3 background is very low, with only ~7% of the chains transferred to protein containing the normal glucotriose unit (Verostek et al., 1993a). In contrast, alg9 yeast transfer a fully glucosylated Glc 3 Man 6 GlcNAc 2 to protein, although little or no Glc 3 Man 6 GlcNAc 2 -PP-Dol accumulates in the OSL pool (Cipollo and Trimble, 2000; Burda et al., 1999). In some yeasts carrying the alg12 deletion, a small amount of Glc 3 Man 7 GlcNAc 2 - PP-Dol accumulates (Burda et al., 1999), but it is clear from Figure 6 that Glc 3 Man 7 GlcNAc 2 is the primary glycan transferred to protein in alg12 yeast. Overexpression of ALG6 in alg3, alg9, andalg12 yeasts increases the level of fully glucosylated OSL and, in the case of alg3 and alg9, the level of glycosylation site occupancy on CPY (Burda et al., 1999). This suggests that the addition of the first Glc residue by Alg6p is the rate-limiting step for full glucosylation, but that once fully glucosylated, even truncated manno-lipids can serve as good substrates for OST. Thus the enhanced glycosylation of invertase observed in alg12 cells relative to that seen in alg9 (Cipollo and Trimble, 2002) is not due to increased OST function stimulated by the presence of the added α1,2-linked central-arm residue 10 but rather to that residue s capacity to promote full glucosylation by potentiation of Alg6p activity. Only a small residual amount of Glc remains on glycans from alg12 invertase, and it is clear from the [2-3 H]Man pulse-chase study (Figure 6) that the glucotriose unit is both efficiently added to OSL and trimmed from glycoproteins in the ER. Alg9 cells also efficiently trim Glc residues and retain a similar amount of residue G 1 to that seen in alg12 cells (Cipollo and Trimble, 2000), as do wild-type cells on wholecell N-glycans (Trimble and Atkinson, 1992). This indicates that in the alg12 background, the upper arm residues 6 and 9 (Scheme 1) are not major structural determinants for the activities of either ER glucosidases I or II, nor does the retention of residue 10 in alg impair glucosidase trimming. Trimming in the ER of the glucotriose unit and α1,2-linked Man residue 10 (Scheme 1A) appears to act as a biological timer for protein maturation in yeast (Jakob et al., 1998), as well as in higher eukaryotes (Helenius et al., 1997; Chung et al., 2000). Alg12 iscompromisedinitsabilitytoremove misfolded CPY (designated CPY*) from the ER via ER-associated degradation, implying that failure to remove α1,2-linked Man 10 extends the time in which a misfolded protein is tolerated in the ER before degradation (Jakob et al., 1998). It is noteworthy in the current work that over 85% of alg12 invertase glycans retained the central-arm α1,2-linked residue 10, demonstrating in vivo that the upper-arm α1,2manα1,6manresidues 6 and 9 (Scheme 1A) are required for optimum Mns1p activity. This confirms earlier in vitro studies that estimated the rate of residue 10 removal in Man 7 GlcNAc-ol structures lacking the upper-arm α1,2manα1,6man- residues 6 and 9 to be only 10% the rate of removal from Man 9 GlcNAc-ol (Ziegler and Trimble, 1991). Mannan outer-chain synthesis begins with the addition of α1,6-man residue 12 to the lower-arm α1,3-man core residue 5, catalyzed by Och1p (Reason et al., 1991) in the cis-golgi 759

12 J.F. Cipollo and R.B. Trimble (Franzusoff and Schekman, 1989; Scheme 1D). The substrate specificity of the Och1p was characterized in vitro using pyridylaminated oligosaccharides (Nakayama et al., 1997). With the alg3 form of Man 5 GlcNAc 2 -PA (Scheme 1B without residue 7) as acceptor, only 9% as much Och1p product was formed as when Man 8 9 GlcNAc 2 -PA was the substrate. By contrast, 60% of a Man 7 GlcNAc 2 -PA, having the structure of the alg9 Man 6 GlcNAc (Scheme 1B) with upper-arm α1,6- Man residue 6 added (Scheme 1A), was elongated by Och1p. This defines core residues 6 and 7 (Scheme 1) as structural determinants for Och1p activity. Although the extent of elongation of Man 8 GlcNAc 2 -PA and Man 9 GlcNAc 2 -PA, which differ only by the presence of the central-arm α1,2-linked residue 10 in the latter (Scheme 1A), was similar, no kinetic data were reported in this study. Thus the rates of α1,6-man addition to Man 8 and Man 9 processing intermediates may differ. In this regard, Puccia and co-workers (1993) reported that [ 35 S]-labeled invertase from mns1 cells migrated slightly faster on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than that of wild type. Furthermore, comparison alg3, alg9, and alg12 glycans from invertase with one hexose added in the Golgi (Hex 6 GlcNAc 2 for alg3, Verostek et al., 1993b; Hex 7 GlcNAc 2 for alg9, Cipollo and Trimble, 2000; and Hex 8 GlcNAc 2 for alg12, current study), shows that 3%, 22%, and 15%, respectively, have the α1,6-man added by Ochlp. Thus in vivo the presence of residue 10 in both the mns1 and alg12 backgrounds appears to hinder Och1p activity to some extent. This is consistent with the observed mild hypoglycosylation of invertase seen in alg12 cells compared to wild-type cells (Cipollo and Trimble, 2002). Materials and methods Bio-Gel P-4 was from Bio-Rad Laboratories. American Radiolabeled Chemicals supplied [2-3 H]Man (20 Ci/mmol). Ecolume scintillation cocktail was purchased from ICN Pharmaceuticals. Sigma was the source for CST and 99.8% and 99.96% D 2 O; % D 2 O was from Cambridge Isotopes Laboratories. Man 3 GlcNAc[ 3 H]ol was from a previous study (Trimble and Atkinson, 1986). Endo H and PNGase F were prepared as described previously (Trumbly et al., 1985; Plummer et al., 1991). All solvents were American Chemical Society reagent grade or better. N-glycans from alg12 external invertase High-specific-activity external invertase was purified from alg12 cells harvested during glucose derepression under growth conditions optimized for low PrAp activity (Cipollo and Trimble, 2002) as described (Verostek et al., 1993a). N-linked oligosaccharides were hydrolyzed from invertase by treatment with PNGase F, isolated by solvent precipitation (Verostek et al., 2000), and then chromatographed on a calibrated column of Bio-Gel P-4 (95 cm 16 mm) with 0.1 N acetic acid/1% 1-butanol as the eluant at 8.8 ml/h at room temperature. Fractions of 0.73 ml were collected, and aliquots were assayed for total hexose and radioactivity from included internal marker(s) of Glc 3 [ 3 H]Man 9 GlcNAc 2 and/or [ 3 H]Man 3 GlcNAc-ol. Glycosidase digestions Endo H and PNGase F digestions followed standard protocols (Tarentino et al., 1989). SDS was removed from the solubilized glycoproteins prior to PNGase F digestion by precipitation with 80% acetone and solubilization in 50 mm sodium phosphate buffer, ph 8.5 (Verostek et al., 2000). MS MALDI/TOF MS was performed on a Bruker Reflex Instrument. Samples of pmol were prepared with 2,5-dihydroxybenzoic acid as matrix. Data accumulated for ns pulses of the 337-nm laser were averaged for each sample. Analyses were performed in linear and reflective mode. HPAEC branch isomer analysis Pooled aliquots of N-glycans were chromatographed on an HPAEC system using a voltage PAD response detector and an analytical (4 250 mm) PA-100 column. Samples were separated using 100 mm NaOH accompanied by the following sodium acetate gradient: isocratic at 35 mm for 5 min, and then mm over 45 min. Individual runs included raffinose or known glycans as internal standards. 1 H NMR spectroscopy Oligosaccharides ( mg) were exchanged with D 2 Oand examined at 300 K and/or 318 K by 1D and 2D DQF COSY phase-sensitive 1 H NMR spectroscopy at 500 MHz as described (Cipollo and Trimble, 2000). Line broadening of 1 2 Hz/Hz was used in both dimensions of 2D DQF COSY experiments for signal enhancement, and a skewed sine-bell weighting function was used in t 2 to reduce dispersive line shape. [2-3 H]Man pulse-chase analysis of N-glycan processing in vivo Alg12 cells were grown overnight to stationary phase in YPD and collected by centrifugation for 5 min at 3000 rpm at room temperature in a Sorvall TC6 centrifuge equipped with an H400 rotor. The yeast were washed twice in glucose-freeyp medium by centrifugation and incubated in the presence or absence of 5 mm CST for 1.5 h in YP + 1% glucose. The yeast cells were again washed twice in glucose-free YP medium by centrifugation; cells ( ) were resuspended to a total volume of 500 µl in YP % Glc containing 500 µci [2-3 H]Man. After 2 min of labeling, a 2000-fold excess of unlabeled Man was added to the reactions. Aliquots of 125 µl were taken at 0, 1, and 10 min of chase, and reactions were terminated by rapid addition to 4 ml of CHCl 3 /CH 3 OH (3:2) while vortexing. The cell pellets were washed and OSL fraction isolated using the method of Zufferey et al. (1995)). Labelled glycans were isolated from the pulse/chase cell pellets by endo H hydrolysis as described (Cipollo et al., 2001). Acknowledgments The collaborative help of the Wadsworth Center Biological Mass Spectrometry and Structural NMR Facility cores is deeply appreciated, as is preparation of this manuscript by Tracy Godfrey. This work was supported in part by U.S. Public Health Service grant GM23900 (to R.B.T.). 760