Birgit Absmanner

Verena Schmeiser

Michael Kämpf

Dr Ludwig Lehle
email: ludwig.lehle@biologie.uni-regensburg.de

Biochemical characterization, membrane association and identification of amino acids essential for function of Alg11 from Saccharomyces cerevisiae, an α1,2-mannosyltransferase, catalyzing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide

Keywords: Protein N-glycosylation, ALG11, dolichol, lipid-linked oligosaccharide, mannosyltransferase, Saccharomyces cerevisiae

    Biochemical characterization, membrane association and identification of amino acids essential for function of Alg11 from Saccharomyces cerevisiae, an α1,2-mannosyltransferase, catalyzing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide

INTRODUCTION

The pathway of N-linked protein glycosylation in eukaryotes is highly conserved and starts with the assembly of the common core oligosaccharide donor Glc 3 Man 9 GlcNAc 2 -PP-Dol, the glycan moiety of which is subsequently transferred by the oligosaccharyltransferase complex onto selected Asn-X-Ser/Thr acceptor sites of the nascent polypeptide chain [START_REF] Helenius | Roles of N-linked glycans in the endoplasmic reticulum[END_REF][START_REF] Lehle | Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases[END_REF]. The initial steps of this multistep pathway up to Man 5 GlcNAc 2 -PP-Dol take place at the cytosolic side of the endoplasmic reticulum (ER), using sugar nucleotides as glycosyl donors [START_REF] Burda | The dolichol pathway of N-linked glycosylation[END_REF]. The heptasaccharide is then translocated across the bilayer into the lumen of the ER by a specific ATP-independent flippase, the molecular and mechanistic details of which remain yet to be elucidated [START_REF] Rush | Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast[END_REF][START_REF] Sanyal | Specific transbilayer translocation of dolichollinked oligosaccharides by an endoplasmic reticulum flippase[END_REF]. Subsequently, Man 5 GlcNAc 2 -PP-Dol is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol, respectively, in an ordered manner based on differences in the substrate specificity of the various glycosyltransferases [START_REF] Burda | Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases[END_REF]. Yeast genetic techniques in the last two decades have opened new approaches to identify genes of the pathway, among those alg mutants (for asparagine-linked glycosylation), isolated in the pioneering work by Robbins and co-workers, were extremely helpful [START_REF] Huffaker | Yeast mutants deficient in protein glycosylation[END_REF]. Most of these mutants were found to be defective in the assembly of the lipid-linked oligosaccharide. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides [START_REF] Beck | Complementing mutant alleles define three loci involved in mannosylation of Man5-GlcNAc2-P-Pdolichol in Chinese hamster ovary cells[END_REF][START_REF] Hall | Reduced utilization of Man5GlcNAc2-P-P-lipid in a Lec9 mutant of Chinese hamster ovary cells: analysis of the steps in oligosaccharide-lipid assembly[END_REF][START_REF] Zeng | Isolation of Chinese hamster ovary cell lines producing Man3GlcNAc2 asparagine-linked glycans[END_REF]. More recent studies of lipid-linked oligosaccharide (LLO) assembly have revealed quite some unexpected findings. The addition of the second GlcNAc residue to form GlcNAc 2 -PP-Dol is catalyzed not by a single subunit enzyme, but by a dimeric complex, in which membrane-bound Alg14 recruits soluble Alg13 to the cytosolic face of the ER to form the active enzyme [START_REF] Bickel | Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Alg13p and Alg14p form a complex required for the formation of GlcNAc(2)-PP-dolichol[END_REF][START_REF] Chantret | Two proteins homologous to the N-and C-terminal domains of the bacterial glycosyltransferase Murg are required for the second step of dolichyl-linked oligosaccharide synthesis in Saccharomyces cerevisiae[END_REF][START_REF] Gao | Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation[END_REF]. Biochemical characterization of Alg2 disclosed it as a bifunctional enzyme required for both the transfer of the α1,3and α1,6-mannose-linked residue to Man 1 GlcNAc 2 -PP-Dol to form Man 2 GlcNAc 2 -We therefore performed a mutational analysis of this motif as well as other amino acids, to identify residues required for Alg11 activity.

EXPERIMENTAL Yeast strains, media, and genetic methods

The following strains were used: SS330 (MATa ade2-101 ura3-52 his3∆200 tyr1), W303-1A (MATa leu2-3 leu2-112, his3-11 his3-15 ura3-1 ade2-1 trp1-1), BAY2402 (MATα ura3-1 his3-11 leu2-3_12, trp1Δ2 ade2-1 can1-100 ALG11::kanΔ MX4), STY50 (MATa his4-401 leu2-3,-112 trp1-1 ura3-52 HOL1-1 SUC2::LEU2). Strains were grown in YPD medium (1% yeast extract, 2% bacto-peptone, 2% glucose) or in selective YNB medium (0.67% YNB, 0.5% casamino acids, 2% glucose) supplemented with amino acids and nucleotide bases, as required. In the case of Δalg11 the medium contained, if not indicated otherwise, also 0.5 M KCl.

To construct the expression plasmids pVT100-Alg11-ZZ and pVT100-Alg11-Bio, genomic ALG11 was amplified from W303-1A by PCR and engineered with HindIII and BamHI restriction sites at the 5' and 3' ends, respectively, for cloning purpose, and ligated into the HindIII / BamHI cut vectors pVT100-ZZ and pVT100-Bio, respectively, placing ALG11 under the control of the constitutive ADH1 promoter. The constructs were sequenced and the functional expression of Alg11 containing two Protein A (ZZ) epitopes or the Bio-epitope in frame at the C-terminus were verified by immunoblotting and complementation of the growth phenotype of the Δalg11 mutant. For PCR amplification of ALG11-ZZ the following primers were used: ALG11 fw (5'-CCCAAGCTTAAAATGGGCAGTGCTTGGACAAACTAC-3') and ALG11 rev (5'-CGGGATCCGCCCCTTTCCTCCTCTTC-3'). To generate the truncated Alg11-Bio reporter variants, internal ALG11 specific reverse primers, annealing at the positions as indicated in the corresponding figures, were used together with the above ALG11 fw primer (primer sequences can be received on request). For constructing the Alg11-Suc2/His4C topology reporter, plasmid pJK90 was used to engineer the ALG11 variants into the SmaI restriction site and the constructs were transformed into STY50 for analysis. To generate site specific alg11 mutants, the Quick Change ® II XL Site-Directed Mutagenesis Kit (Stratagene) was used according to the manufacturer's instructions. Transformation into yeast and E.coli was carried out using standard techniques.

In vivo labelling of lipid-linked oligosaccharides with [ 3 H]mannose

Labelling with [2-3 H]mannose (15 Ci/mmol; GE Healthcare) and analysis of lipid-linked oligosaccharides by HPLC was performed as described previously [START_REF] Knauer | The oligosaccharyltransferase complex from Saccharomyces cerevisiae. Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex[END_REF]. Cells were grown overnight at 25 °C. Subsequently cells were labelled at 25 °C (Δalg11), or at 30 °C (wildtype) as indicated.

Isolation of microsomal membranes and preparation of solubilized enzyme extract

Rough microsomal membranes were isolated as described [START_REF] Knauer | The N-oligosaccharyltransferase complex from yeast[END_REF]. Membranes were resuspended in 30 mM Tris/HCl buffer, pH 7.5, containing 3 mM MgCl 2 , 1 mM DTT, 35% glycerol (v/v) at a concentration of 10 mg/ml protein. For membrane solubilisation, Nonidet P-40 was added to a final concentration of 1%. After 20 min incubation on ice the solubilized extract was separated from insoluble material by centrifugation at 150 000 g for 40 min. All steps were carried out at 4 °C, unless indicated otherwise.

Mannosyltransferase assays

Assay I: Activity of solubilized enzyme was determined with Man 3 [ 14 C]GlcNAc 2 -PP-Dol or [ 14 C]GlcNAc 2 -PP-Dol as acceptor and non-radioactive GDP-Man as glycosyl donor. The reaction contained the following in a final volume of 0.06 ml: 14 mM Mes, pH 6.0, 0.1% Nonidet-P40, 10 mM MgCl 2, 0.1 mM Na-EDTA, 1 mM DDT, 4 mM potassium-citrate, 1 M sucrose, [ 14 C]GlcNAc 2 -PP-Dol (3000 cpm) or Man 3 [ 14 C]GlcNAc 2 -PP-Dol (3000 cpm), 1 mM GDP-Man and solubilized enzyme (equivalent to 70 µg membrane protein). The reaction was started by addition of GDP-Man and incubated at 26 °C, stopped with chloroform/methanol (3:2) at the times indicated. Labelled glycolipids were then extracted, washed and oligosaccharides were released from the dolichol-PP moiety by mild acid and analyzed by HPLC as described [START_REF] Sharma | Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase[END_REF].

RESULTS

Characterization of Δalg11 null mutant

Originally, an alg11 mutant has been isolated by its resistance to sodium vanadate [START_REF] Cipollo | The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum[END_REF]. This selection procedure has been described earlier for the isolation of yeast glycosylation mutants [START_REF] Ballou | Vanadate-resistant yeast mutants are defective in protein glycosylation[END_REF], although the biochemical basis for this is not understood. alg11 was shown to be temperature sensitive and defective in N-glycosylation. This study also implicated that Alg11 is involved merely in the addition of the terminal α1,2-linked mannose to Man 4 GlcNAc 2 -PP-Dol, but enzymatic validation was lacking [START_REF] Cipollo | The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum[END_REF]. To characterize Alg11 in more detail and in particular to define its specific role in LLO synthesis, an Δalg11 null allele has been isolated by sporulation of an ALG11/Δalg11 diploid strain. As we expected that deletion of a gene involved in an early stage of LLO assembly may be lethal or of poor growth, an osmotic stabilizer like sorbitol or KCl was added during the sporulation procedure, to compensate for a weakened cell wall due to defective glycosylation. In fact, sporulation resulted in a 2:2 segregation with two efficiently growing wild-type cells and two slowly growing Δalg11 null alleles (data not shown). In Figure 1A and B the growth behaviour of Δalg11 is depicted in dependency of temperature, osmotic stabilizer and agents interfering with cell wall biogenesis, such as Calcofluor White (CFW) or caffeine. Compared to wild-type cells, Δalg11 grew poorly and osmotic stabilization by KCl only slightly improved growth (Figure 1B). Nevertheless, KCl was added throughout this study. However, deletion of ALG11 caused a temperature sensitive lethality between 32° C and 36 °C (Figure 1A). Likewise addition of 5 µg/ml CFW or 5 mM caffeine to the medium abolished completely growth of cells. CFW binds to chitin and interferes with cell wall assembly [START_REF] Ram | A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white[END_REF], whereas the phosphodiesterase inhibitor caffeine is thought to exert its effect via protein kinase C, a central element in the regulation of cell wall integrity [START_REF] Levin | The proliferation of MAP kinase signaling pathways in yeast[END_REF]. Figure 1C shows the analysis of lipid-linked oligosaccharides of cells metabolically labelled with [ 3 H]mannose. Whereas in wild-type yeast the fully assembled core oligosaccharide Glc 3 Man 9 GlcNAc 2 -PP-Dol and various intermediates occurred, in Δalg11 the largest oligosaccharide had a composition of Man 7 GlcNAc 2 -PP-Dol. According to the results described below, demonstrating that ALG11 encodes the mannosyltransferase catalyzing the formation of both Man 4 GlcNAc 2 -PP-Dol and Man 5 GlcNAc 2 -PP-Dol, one would have expected Man 3 GlcNAc 2 -PP-Dol as the largest oligosaccharide in the deletion mutant. As shown earlier Man 7 GlcNAc 2 -PP-Dol in Δalg11 is a isomeric species structurally different from the normal biosynthetic LLO-Man 7 of the wildtype arising by elongation of the α1,6-arm by luminal mannosyltransferases with Man-P-Dol as glycosyl donor [START_REF] Cipollo | The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum[END_REF]. However, these shortened and aberrant glycan chains are non optimal substrates for OST and are less efficiently transferred to the protein, as can be seen from the analysis of the glycosylation status of the vacuolar model glycoprotein carboxypeptidase CPY (Figure 1D) (see also further discussion below). CPY from wild-type cells contains four Nlinked glycan chains and migrates as a distinct band, whereas in the Δalg11 strain the glycosylation defect gives rise to CPY forms possessing a higher mobility on SDS-gels. Complementation with a plasmid harbouring ALG11 or a variant with a C-terminal Protein A (ZZ)-epitope for detection (see below) restored wild-type glycosylation. This result also indicates that the epitope does not disturb Alg11 activity.

Alg11 is a mannosyl transferase with dual function catalyzing both the formation of Man 4 GlcNAc 2 -PP-Dol and Man 5 GlcNAc 2 -PP-Dol

To unravel the mannosylation defect of Δalg11 in more detail, we first established an enzymatic test that allowed to measure early reactions of LLO synthesis from GlcNAc 2 -PP-Dol to Man 5 GlcNAc 2 -PP-Dol involving the participation of Alg1, Alg2 and Alg11 in that order. As can be seen in Figure 2A, incubation of a detergent solubilized enzyme extract from microsomal membranes of wild-type yeast with radioactive [ 14 C]GlcNAc 2 -PP-Dol as acceptor and unlabeled GDP-Man as glycosyl donor led to elongation of the acceptor oligosaccharide up to Man 5 GlcNAc 2 -PP-Dol (left and middle panel). In contrast, an extract from Δalg11 elongated only to Man 3 GlcNAc 2 -PP-Dol, indicating that Alg11 catalyzes at least the transfer of the fourth mannose (right panel). Prevention of further elongation beyond Man 5 GlcNAc 2 -PP-Dol in the detergent extract from wild-type, as compared to in vivo, could be due to the fact that the proper reaction conditions are not given, no Dol-P-Man is generated, or the respective transferases are not solubilized or are inactive.

In order to measure exclusively Alg11 activity, we introduced a protein A (ZZ)-epitope at the C-terminus of plasmid borne ALG11, allowing to immunoprecipitate Alg11-ZZ from the solubilized extract with IgG-Sepharose as affinity matrix. The ALG11-ZZ plasmid and as control a plasmid containing ALG11 without the ZZ-epitope were transformed into Δalg11 and the enzyme activity was determined. In the experiment depicted in Figure 2B Man 3 [ 14 C]GlcNAc 2 -PP-Dol was converted to Man 5 [ 14 C]GlcNAc 2 -PP-Dol (middle panel). But no transfer onto Man 3 [ 14 C]GlcNAc 2 -PP-Dol was measureable in the control experiment, in which solubilized extract from membranes of Δalg11 cells expressing the non-tagged Alg11 was incubated with IgG-Sepharose beads (right panel). This means that immunoprecipitation of Alg11 is specific, rather than Alg11 binds non-specificly to the affinity matrix. Assuming that no other interacting protein is pulled down, because of extensive washing of the immunoprecipitate applying detergent, the above results demonstrate that Alg11 is able not only to catalyze the addition of the fourth, but also of the fifth, α1,2-linked mannose residue.

Investigation of the membrane integration of Alg11 reveals only one membrane spanning domain

Alg11 is a protein with a calculated molecular mass of 63 kDa consisting of 548 amino acids. A hydropathy plot reveals three hydrophobic domains (Figure 3A). Topology predictions suggest either three transmembrane spanning helices (with TMpred and TopPred) or only one helix (with TMHMM and SOSUI). From this and taking into account that the reactions catalyzed by Alg11 must occur at the cytoplasmic face of the ER, because the nucleotide sugar donor is only present in the cytosol, three topology models (model A, B and C) can be proposed as shown in Figure 3B. As Alg11 belongs to the GT4 glycosyltransferase family and therefore should present a GT-B fold consisting of two Rossmann domains, models A and B requiring around 300 amino acids can most probably excluded. Nevertheless, to experimentally prove or disprove these predictions, we used two approaches. We designed various fusions between Alg11 and a Bio-tag, or Alg11 and a Suc2/His4C-tag as topologysensitive reporters (Figure 3C). In case of the Bio-tag, this domain is a substrate for biotin ligase [START_REF] Stolz | Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation[END_REF] and will be biotinylated only, when oriented to the cytosolic side, as this reaction takes place exclusively in the cytosol. His4C maintains histidinol dehydrogenase activity, the last step in histidine biosynthesis, and his4 mutants, expressing a Suc2/His4C fusion, are able to grow on minimal histidinol medium only, when the His4C domain is at the cytoplasmic side of the ER, but not when targeted to the ER lumen. In addition, in the latter case the chimeric protein becomes extensively glycosylated due to the presence of N-glycosylation sites [START_REF] Kim | Determination of the membrane topology of Ost4p and its subunit interactions in the oligosaccharyltransferase complex in Saccharomyces cerevisiae[END_REF][START_REF] Sengstag | Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase[END_REF]. As shown in Figure 3D, lanes 2-5, Alg11 fusions with the Bio-tag engineered C-terminal to the postulated first (Alg11 1-139 ), second (Alg11 1-396 ) and third (Alg11 1-548 ) transmembrane domain (TMD) were all biotinylated, indicating that these fusions were oriented to the cytosol. These results exclude model A and B with three TMD and would favour model C with only one TMD. Such a topology is also in agreement with the HisC4 technique approach. All fusions supported growth on histidinol, implying that the C-terminus of the respective constructs localized to the cytosol (Figure 3E). Moreover, the topology of the constructs has been validated by analyzing their glycosylation status. Compared to a control glycoprotein (Figure 3F, lanes 7 and 8), treatment with endoH indicated that all fusions were resistant to enzyme treatment and thus were not glycosylated, and the respective C-terminus must be cytosolic orientated therefore. In order to localize the N-terminus and also to demonstrate that the first hydrophobic segment acts indeed as a transmembrane spanning segment, we also made a construct with the Bio-tag at the N-terminus. As can be seen in Figure 3D, lane 1, this fusion is not biotinylated, indicating ER luminal orientation of the Nterminus. As will be demonstrated below (Figure 4), the middle and the C-terminal hydrophobic regions, although not functioning as TMDs, nevertheless contribute to anchoring Alg11 to the ER in a non-peripheral manner, presumably by integrating into the outer leaflet of the ER membrane. Therefore, model D is proposed for the membrane integration of Alg11.

The N-terminal transmembrane domain is not essential for Alg11 function

Experiments described above demonstrate that Alg11 consists of only one TMD. Therefore, we asked, which function this TMD as well as the two other predicted, hydrophobic segments could serve. Several Alg11 truncations with a C-terminal ZZ-epitope for detection purpose, as illustrated in Figure 4A, were engineered and their role was investigated with respect to growth, N-glycosylation, membrane anchoring and enzyme activity. The constructs were cloned into a constitutive expression vector and analyzed in the Δalg11 strain. Quite unexpected, deletion of the N-terminal TMD in the Alg11 45-548 variant led to a functionally active mannosyltransferase, which supported the ts-growth defect of Δalg11 (Figure 4D), rescued the underglycosylation of CPY (Figure 4E, compare lanes 1 and 2) and was also 4F). As shown by Western blot analysis (Figure 4B, lane 4) the TMD lacking construct still bound to the membrane, indicating that one or both of the two other hydrophobic domains contribute to membrane anchoring as well, even though not in a transmembrane spanning manner.

To address the membrane association more specifically, we performed an alkaline sodium carbonate extraction of membranes, known to release peripheral attached proteins, and compared it with a 2% NP40 or 0.2% SDS detergent treatment. As shown in Figure 4C, there was no significant difference in extraction between the full length Alg11 1-548 and the Nterminal truncated Alg11 45-548 variant. Even extraction with 2% detergent caused only partial solubilisation of the N-terminally truncated version, to a similar extent as in the case of wildtype Alg11. These results clearly do not suggest a peripheral attachment of truncated Alg11 lacking the N-terminal TMD. Hence, together with the results from the Bio-and His4localization experiments, a topology is proposed as depicted in model D (Figure 3), positioning the hydrophobic helices into the outer leaflet of the membrane (for further discussion see below).

In contrast to N-terminal truncation, C-terminal deletions were detrimental for Alg11 activity. Removal of the third hydrophobic domain in the Alg11 1-418 construct led to underglycosylation of CPY similar to the vector control (Figure 4E, lanes 1 and 4), but still allowed some residual cell growth (Figure 4D). However, marginally further truncation removing the conserved EX 7 E motif, as is the case in the Alg11 1-396 variant, resulted in a complete loss of Alg11 function.

Analysis of Alg11 activity by site specific mutagenesis

To identify amino acids important for catalysis and function of Alg11, we performed site specific mutagenesis in conserved amino acid residues and motifs, respectively, discussed in the functional context of glycosyltransferases. As already mentioned, a variety of Pfam GT1F glycosyltransferases contain the C-terminal signature sequence motif EX 7 E [START_REF] Bourne | Glycoside hydrolases and glycosyltransferases: families and functional modules[END_REF]. The two glutamate residues of the motif have been proposed to be involved in catalysis, even though their contributions have been discussed controversial and need to be identified by structural analysis [31][START_REF] Cid | Identification of two essential glutamic acid residues in glycogen synthase[END_REF][START_REF] Kostova | Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly[END_REF]. Figure 5B shows an alignment of the respective region of Alg11 and Alg2 mannosyltransferases from S. cerevisiae that are closely related and are grouped into family 4 of the CaZY database. In addition also the human Alg11 and AceA, a mannosyltransferase from Acetobacter xylinum, are aligned. We replaced both E405 and E413 by alanine and found that mutating the first Glu (E405) caused an underglycosylation of CPY, whereas altering the second Glu (E413) had no effect (Figure 5B, lanes 2 and 7). This is in agreement with the hypothesis that the first Glu of the EX 7 E motif is more critical for activity [31] and is also in accord with the Alg11 enzyme activity, expressed in E. coli [START_REF] O'reilly | In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis[END_REF]. Interestingly, the reduced activity of the E405A mutation was not sufficient to prevent cell growth. This variant was still able to complement the Δalg11 growth deficiency at 36 °C (Figure 5A). Also three other selected mutations within the motif, such as H406A, F407A or V412A, were not detrimental for growth, albeit impairment of glycosylation of CPY was observed in the first two mutants. However, an E405A/E413 as well as an E405/H406A double mutant no longer supported growth, indicating that further constraints of E405 are deleterious. The loss of activity is not caused by a failure of Alg11 expression, as proven by Western blot analysis (data not shown).

In addition we mutated several lysine and glutamate residues to alanine, proposed to be involved in nucleotide sugar donor binding of glycosyltransferases [31]. From six conserved mutations (see Table I), K302, E318, K319, K322, K343 and E356, only K319 (which is also conserved in AceA) was detrimental and caused loss of Alg11 activity, as revealed from the analysis of cell growth (Figure 5D) and glycosylation of CPY (Figure 5C). The significance of residue K319 is also emphasized by the fact that amino acid exchanges in the immediate adjacent amino acids, E318 or K322, had no effect. Similarly, mutating various aspartate residues as potential catalytic base had no effect.

Finally, a conserved glycine rich motif in the N-terminal region was investigated (Figure 5 E and F) that may be part of a flexible loop important for catalysis of glycosyltransferases both in the GT-A and GT-B families [START_REF] Breton | Structures and mechanisms of glycosyltransferases[END_REF][34][35][START_REF] Wrabl | Homology between O-linked GlcNAc transferases and proteins of the glycogen phosphorylase superfamily[END_REF]. Replacing G84, G85 or G87 by proline residue that may eventually lead to structural constraints, was found to be crucial for activity for G85 and G87, whereas a conservative substitution by alanine had no effect on CPY glycosylation and growth. Table I compiles the various mutations.

DISCUSSION

In this report we further characterized Alg11 from Saccharomyces cerevisiae as a mannosyltransferase with a dual function in the early phase of lipid-linked oligosaccharide assembly (Figure 6), investigated its membrane association as well as amino acid residues, important for Alg11 function. The results close a gap in the identification of the last, ill defined glycosyltransferase of the dolichol mediated reactions of N-linked glycosylation. As this pathway is highly conserved from yeast to human, the results may be of general relevance and one can expect deficiencies of the Alg11 enzyme as cause for a new, fatal type of Congenital Disorders of Glycosylation (CDG), as it applies for other defects in lipid-linked oligosaccharide biosynthesis [START_REF] Lehle | Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases[END_REF]. We first established an enzymatic in vitro test for Alg11 transferase and demonstrate that membranes from wild-type cells, or an Alg11 immunoprecipitate from a solubilized membrane extract, catalyze the transfer of two mannose residues from the GDP-Man donor to Man 3 GlcNAc 2 -PP-Dol giving rise to Man 5 GlcNAc 2 -PP-Dol as final product. These reactions were not taking place, when membranes from Δalg11 cells were used as enzyme source. This result clearly demonstrates that Alg11 catalyzes both final glycosylation steps on the cytosolic side of the ER. Bifunctionality of glycosyltransferases is rather rare but not without precedent. It has also been observed for the Alg9 mannosyltransferase adding the terminal seventh and ninth, each α1,2-linked, mannose residue during Man 9 GlcNAc 2 -PP-Dol synthesis [START_REF] Frank | ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis[END_REF]. In case of the Pgl pathway of protein Nglycosylation in the bacteria Campylobacter jejuni, having similarities to the dolichol pathway, the PglH transferase adds iteratively three terminal 1,4-linked GalNAc residues to an undecaprenyl diphosphate-linked heptasaccharide [START_REF] Troutman | Campylobacter jejuni PglH is a Single Active Site Processive Polymerase that Utilizes Product Inhibition to Limit Sequential Glycosyl Transfer Reactions[END_REF]. Whereas in both examples the same type of linkage is catalyzed, in case of Alg2, acting on the formation of the two mannose additions in LLO-synthesis prior to Alg11, bifunctionality comprises even the formation of two different linkages, an α1,3and α1,6-bond [START_REF] O'reilly | In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis[END_REF]15]. Deletion of ALG11 was not lethal, but led to a severe, slow growth phenotype. However, additional temperature stress or stress challenged by compounds interfering with cell wall formation caused lethality in Δalg11 (Figure 1). This phenotype is in stark contrast to glycosylation defects affecting luminal steps of LLO synthesis that do not display growth impairments, unless in combination with other defects, e.g. with defects in OST or in the glucosylation of LLO [START_REF] Stagljar | New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus[END_REF]. On the protein level Δalg11 displays a severe hypoglycosylation of proteins. In principle this could be attributed, on account the genetic defect of the mutant, to inefficient flipping of the Man 3 GlcNAc 2 -PP-Dol and/or reduced transfer to the nascent polypeptide of this or the aberrant Man 6/7 -GlcNAc 2 -PP-Dol isomers, generated through luminal action or Man-P-Dol dependent glycosyltransferases. It is known that abnormal and truncated LLOs are poor substrates for OST, leading to reduced glycan transfer to the nascent polypeptide chain. However, it was recently shown that in reconstituted proteoliposomes Man 3 GlcNAc 2 -PP-Dol is flipped almost as well as Man 5 GlcNAc 2 -PP-Dol [START_REF] Sanyal | Specific transbilayer translocation of dolichollinked oligosaccharides by an endoplasmic reticulum flippase[END_REF]. Whether this holds also true in vivo is not known. It has been found that overexpression of RFT1 in Δalg11, an integral, essential membrane protein, originally proposed to be the flippase, what, however, is no longer tenable on account of recent results, increased the yield of Man 7 GlcNAc 2 -PP-Dol and diminished the growth defect of the cells and hence Rft1 may contribute to controlling the access of dolichol-linked oligosaccharides to the flippase. It should be emphasized that our analysis does not allow distinguishing, which of the truncated oligosaccharide chains are transferred. But analysis of glycans of the total mannoprotein fraction revealed that the Man 7 GlcNAc 2 isomer structure is detectable in Δalg11 [START_REF] Cipollo | The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum[END_REF]. Worth mentioning in the context of flipping is also the finding that disruption of ALG2 was shown to be lethal in S. cerevisiae. This gene catalyzes the two previous glycosylation steps prior to Alg11 in LLO biosynthesis (Figure 6) and its disruption results in the accumulation of Man 1 GlcNAc 2 -PP-Dol. Thus one may speculate that glycan chains smaller than Man 3 GlcNAc 2 may no longer be compatible with viability of bakers yeast, because of insufficient flipping or other reasons. On the other hand in an alg2 null mutant of the zygomycete Rhizomucor pusillus, compared to bakers yeast a slowly growing fungi, Man 1 GlcNAc 2 -PP-Dol was the largest oligosaccharide synthesized and transferred to the protein, and yet the mutant was still viable [START_REF] Takeuchi | Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus[END_REF]. Whether this could be due to differences in growth rates and/or the process of flipping is speculative at the moment and remains to be demonstrated.

As mentioned, Alg11 belongs to glycosyltransferase family 4 (CAZY) and glycosyltransferase 1 family (Pfam), respectively. In many of the retaining transferases an EX 7 E motif can be detected in the C-terminal region, as it is also true for Alg11. These conserved acidic amino acids have been suggested to be involved in catalysis as a nucleophile, stabilizing the donor substrate [31- [START_REF] Kostova | Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly[END_REF][START_REF] Troutman | Campylobacter jejuni PglH is a Single Active Site Processive Polymerase that Utilizes Product Inhibition to Limit Sequential Glycosyl Transfer Reactions[END_REF][START_REF] Guerin | Molecular recognition and interfacial catalysis by the essential phosphatidylinositol mannosyltransferase PimA from mycobacteria[END_REF][START_REF] Muniz | Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa[END_REF] but with mixed results. As depicted in Figure 5 exchange of E405 in this motif to alanine caused some underglycosylation of CPY but with only a mild effect on cell growth. In contrast, mutation of the second E413 did not alter Alg11 function at all. This emphasis that the first glutamate residue in the motif is more crucial [31]. However, mutating both E405 and E413 residues to alanine, caused lethality. As also an E405/H406 double mutant was not compatible with living of cells, whereas the single mutation of H406 to alanine was not detrimental, this may indicate that additional perturbation in the local structure of the motif by a further mutation is not restricted to the second glutamate. In this context it is worth mentioning that also in the case of Alg2 it was shown that single mutations of the glutamic acid residues in the EX 7 E motif do not disturb enzyme activity or cell viability [15].

In addition we detected Lys319, also conserved in Alg11 from other species as well as in AceA mannosyltransferase, as an important amino acid for Alg11 activity (Figure 5C andD). In the case of AceA this lysine residue has been proposed to function in nucleotide sugar binding [31]. Substitution of Lys319 by alanine in Alg11 led to underglycosylation as well as to slower growth and eventually to loss of viability upon stress provoked by caffeine (Figure 5D). Likewise conserved aspartate residues were mutagenized, which have also been discussed as potential nucleophiles by deprotonating the hydroxyl group of the acceptor, acting as catalytic nucleophilic base attacking the C1 atom of the glycosyl donor. However, none of these residues are required for Alg11 function (Figure 5C and Table 1).

Another severe functional influence was observed exchanging glycine in a glycine rich domain that could be part of a potential flexible glycine loop. Studies on several glycosyltransferases both from the GT-A and GT-B superfamily have shown that one or two flexible loops at the substrate binding site of the enzyme undergo marked conformational changes from an open to a closed conformation, in which the loop acts as a lid covering the bound glycosyl donor [START_REF] Breton | Structures and mechanisms of glycosyltransferases[END_REF]34,35]. We have exchanged glycine residues 84, 85 and 87 of a conserved region both by alanine and by proline. In the later case the replacement is expected to have a strong effect on the protein architecture. Whereas substitution by alanine did not affect glycosylation and cell growth, the G84P and G87P variant was critical and caused loss of Alg11 function. The significance of these mutations is underscored by the fact that a G85P mutation was not detrimental. Nevertheless, a final evaluation of the mutational analysis allowing a mechanistic explanation needs support by crystal structure information. We also cannot infer from our analysis, whether the different mutants select for transfer of different glycan structures as well as whether Man 4 GlcNAc 2 -PP-dolichol formation can be uncoupled of Man 5 GlcNAc 2 -PP-dolichol.

Another important issue of the study was the examination of the membrane integration of Alg11, which has not been analyzed before. Using two different experimental topologysensitive reporter fusion approaches, we proved that Alg11 consists of only one transmembrane spanning domain with the N-terminus in the lumen of the ER, while the two other predicted hydrophobic sequences in the middle and the C-terminus of the protein do not span the membrane, but seem to insert into the outer leaflet of the membrane as proposed in model D (Figure 3). This conclusion is based on the fact that an N-terminal truncated Alg11 45- 548 variant, lacking the TMD, was tightly integrated into the membrane rather than being peripherally associated upon treatment with alkaline carbonate or neutral detergent (Figure 4C), and furthermore on the observation that the second and third hydrophobic helix are accessible from the cytosolic face (Figure 3). Although an enormous progress has been made to predict structural properties of proteins, there is still not much known about the molecular details of how membrane helices are recognized and integrate into the membrane in the correct way. Membrane-embedded helices can be long or short, cross the membrane at oblique angles, lie flat on the surface, or even span only part of the bilayer and turn back [START_REF] Von Heijne | Membrane-protein topology[END_REF]. Our experimental results do not allow defining the exact mode of integration of the two hydrophobic, non-transmembrane domains. A helical wheel projection of membrane segments 2 and 3 (data not shown) reveals a concentration of hydrophobic and of more polar amino acid residues on two different sites of the helix. This may suggest an interaction with the surface and the non-polar interior of the membrane as discussed for Ost2 [44], a subunit of the OST, or the Sec61α-and Sec61γ-subunit of the translocon [45,[START_REF] Wilkinson | Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex[END_REF]. Moreover, insertion of monotopic proteins is often accomplished by hydrophobic helices interacting with one leaflet of the membrane [START_REF] Bracey | Structural adaptations in a membrane enzyme that terminates endocannabinoid signaling[END_REF][START_REF] Partridge | A transmembrane segment mimic derived from Escherichia coli diacylglycerol kinase inhibits protein activity[END_REF][START_REF] Zakharov | Insertion intermediates of pore-forming colicins in membrane two-dimensional space[END_REF]. Surprisingly, removal of the N-terminal TMD did not disturb Alg11 function in vivo and in vitro as well as its localization (Figure 4D-F). However, C-terminal truncations were detrimental. Whereas deletion of the third hydrophobic domain slightly downstream of the EX 7 E motif (Alg11 1-418 ) caused an underglycosylation and a temperature growth phenotype (Figure 4D andE), further truncation resulting in its removal (Alg 1-396 ) led to an inactive transferase, which was still associated with the ER membrane. The specific role of the N-terminal TMD remains obscure and only speculative for the moment. One possibility is that it contributes, even though not being essential, to interact with other Alg proteins. Thus it has been demonstrated that there exist physical interactions between Alg1, Alg2 and Alg11 [START_REF] Gao | Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum[END_REF]. Further work will also be necessary to understand the mechanistic details of membrane integration as well as the mechanism, how Alg11 transferase adds sequentially two mannose residues. Potentially it is conceivable that it acts in a processive manner, in which the Man 4 GlcNAc 2 -PP-Dol intermediate does not dissociate from the enzyme, or alternatively the intermediate freely dissociates and reassociates as substrate.

Table 1 Site specific mutagenesis of Alg11

Table 1 summarizes the results of site specific mutagenesis. To test the complementation of the Δalg11 growth defect, cells were transformed with mutated ALG11 and incubated at non permissive temperatures as specified in the various figures. For the analysis of the glycosylation defect, the glycosylation status of carboxypeptidase CPY was analyzed by Western blot. In panels five and six the conservation of the mutated amino acids is summarized comparing Alg11 from yeast and human, and Alg11 and Alg2 from yeast, respectively; c, conserved amino acids. The E88A exchange is not documented in Fig. 5. A c c e p t e d M a n u s c r i p t

Growth phenotype and glycosylation defect of Alg11 mutations and conservation of amino acids

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. transferase activity in immunoprecipitates (Ipp). Alg11 with a C-terminal protein A (ZZ)epitope was immunoprecipitated with IgG-Sepharose beads from solubilized extract and the enzyme activity was determined using assay II (left and middle panel). As control, an extract from wild-type cells expressing non-tagged Alg11 was immunoprecipitated, where no elongation tool place (right panel). The positions of standards are indicated, M 1-5 , GlcNAc 2 Man 1-5 and GN 2 , GlcNAc 2 .

Figure 3 Investigation of the membrane integration of Alg11

(A) Hydrophobicity probability plot of Alg11 generated using the TopPred server. (B) Models for the Alg11 membrane topology. The predicted topology of Alg11 suggests three TMD (model A and B); the experimental data using truncated Alg11-reporter fusions and truncated Alg11 variants (see Figure 4) suggest model D, containing one TMD at the N-terminus with orientation to the ER lumen and two additional C-terminal hydrophobic regions that integrate, but do not cross the membrane. Model C is excluded on account of the extraction behaviour of Alg11. (C) Schematic representation of Alg11-reporter constructs. Either a Bio-tag or a SUC2::HisC4-tag was fused in frame at the C-terminal side following amino acids 139, 396 or 548, or in case of the Bio-epitope, also an N-terminal Bio-Alg11 1-548 variant; drawings are not to scale. (D) Analysis of Alg11 topology by Alg11-Bio fusions. Membranes from cells expressing plasmid borne Alg11 constructs, as indicated, were analyzed by Western blotting. Note that in lane 1 the Bio-tag is at the N-terminus (Bio-Alg11), whereas all the other construct are C-terminally tagged (Alg11-Bio). The asterisks indicate biotinylated, endogenous yeast proteins. Note that Alg11 1-396 (lane 3) has the same mobility as one of the endogenous proteins. (E) Analysis of topology by Alg11-Suc2::HisC4 fusions. Strain STY50, containing a his4 mutation, was transformed with truncated Alg11 variants as indicated, and analyzed for growth on histidine or histidinol. Only cells with a cytosolic orientation of the epitope are able to grow on histidinol. (F) Analysis of glycosylation of Alg11-Suc2::HisC4 constructs. Membranes from cells, expressing the various Alg11-Suc2::HisC4 constructs (lanes 1-6), were isolated and incubated without (-) or with (+) endoH for 3 h to release the glycan chains and analyzed by Western blotting. In lanes 7 and 8 an Ost2 1-72 fusion, which is glycosylated, was digested as a control. Constructs were probed with an antibody against the hemagglutinin epitope of the fusion. Pathway of early lipid-linked oligosaccharide formation on the cytosolic side of the ER membrane Biosynthesis starts with the transfer of a GlcNAc-phosphate to dolichol phosphate with formation of the pyrophosphate bond, catalyzed by Alg7. The second step is catalyzed be the dimeric Alg14 / Alg13 complex, whereby membrane bound Alg14 recruits cytosolic Alg13 to the membrane with formation of the active GlcNAc transferase. Subsequent to the addition of the β1,4-linked mannose catalyzed by Alg1, bifunctional Alg2 adds both the α1,3-and α1,6linked mannose residues. The two final α1,2-mannose residues are transferred by Alg11, before the Man 5 GlcNAc 2 -PP heptasaccharide is translocated across the ER membrane to the lumen through action of an ATP-independent flippase, where further elongation occurs to the A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  [ 14 C]GlcNAc 2 -PP-Dol was synthesized according to [23]; Man 3 [ 14 C]GlcNAc 2 -PP-Dol was prepared enzymaticly by elongating [ 14 C]GlcNAc 2 -PP-Dol to Man 3 [ 14 C]GlcNAc 2 -PP-Dol with a solubilized enzyme extract from Δalg11 membranes. Assay II: Determination of enzyme activity in immunoprecipitates of Alg11-ZZ was carried out as follows. 0.2 ml solubilized extract was incubated with 0.1 ml IgG-Sepharose 6 Fast Flow (GE Healthcare) by gently tumbling for 60 min at 4 °C. IgG-Sepharose 6 was equilibrated before use with 40 mM Pipes, pH 6.8, 1% Nonidet-P40, 1 mM DTT and 15% glycerol. After incubation the affinity matrix was washed five times with equilibration buffer and then three times with 20 mM Mes, pH 6, containing 0.1% Nonidet-P40, 1 mM DTT, 10 mM MgCl 2 , 4 mM potassium citrate and 1.5 M sucrose. The pellet was resuspended in 50 µl mannosyltransferase assay I reaction mixture with Man 3 [ 14 C]GlcNAc 2 -PP-Dol as acceptor. The reaction was started by addition of 1 mM GDP-Man (final concentration), incubated for 45 min and processed as for assay I.

  Biochemical Journal Immediate Publication. Published on 24 Nov 2009 as manuscript BJ20091121 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091121 A c c e p t e d M a n u s c r i p t active in vitro using an Alg11 immunoprecipitate to elongate Man 3 GlcNAc 2 -PP-Dol to Man 5 GlcNAc 2 -PP-Dol (Figure

Figure 1

 1 Figure 1 Analysis of the growth and glycosylation defect of Δalg11 (A and B). Growth behaviour of Δalg11 cells. (A) 3 µl of a serial 1:10 dilution of the indicated strains grown in liquid medium were plated on YPD plates containing 0.5 M KCl starting with 3 × 10 4 cells. Plates were incubated at 25, 32, or 36 °C for 72 h. (B) YPD plates were supplemented without additives, with 0.5 M KCl, CFW or caffeine as indicated. (C) Analysis of lipid-linked oligosaccharides in wild-type cells (SS328) and alg11 mutant cells. Lipid-linked oligosaccharides were metabolically labelled for 20 min with [2-3 H]mannose at the temperature indicated, and the oligosaccharides released by mild acid hydrolysis were analyzed by HPLC. The positions of standards are indicated; M 3-8 , GlcNAc 2 Man 3-8 ; G 3 , GlcNAc 2 Man 9 Glc 3 M * 6 and M * 7 denote isomeric forms of GlcNAc 2 Man 7 and GlcNAc 2 Man 8 as illustrated in the pictogram. (D) Western blot analysis of carboxypeptidase Y (CPY). Wildtype (lane1) and Δalg11 cells (lanes 2-4) were cultivated at 25 °C in YPD, containing 0.5 M KCl and the cytosolic fraction, obtained by lysis of cells with glass beads, was analyzed. The equivalent of 10 -7 cells was applied per lane and separated on an 8% SDS-polyacrylamide gel. The position of the mature form of carboxypeptidase Y (mCPY), possessing four N-glycans and of the underglycosylated forms in Δalg11 cells are indicated.

Figure 2

 2 Figure 2 Analysis of Alg11 mannosyltransferase activity(A) Alg11 activity of solubilized enzyme. Membranes from wild-type and Δalg11 cells were solubilized and tested using assay I with [ 14 C]GlcNAc 2 -PP-Dol as glycosyl acceptor as described in the Experimental section. Left panel, incubation without GDP-Man; middle and right panel, incubation with GDP-Man. Lipid-linked oligosaccharides were extracted and the oligosaccharide moiety was released by mild acid and separated by HPLC. (B) Alg11 transferase activity in immunoprecipitates (Ipp). Alg11 with a C-terminal protein A (ZZ)epitope was immunoprecipitated with IgG-Sepharose beads from solubilized extract and the enzyme activity was determined using assay II (left and middle panel). As control, an extract from wild-type cells expressing non-tagged Alg11 was immunoprecipitated, where no elongation tool place (right panel). The positions of standards are indicated, M 1-5 , GlcNAc 2 Man 1-5 and GN 2 , GlcNAc 2 .

Figure 4

 4 Figure 4 Alg11 protein lacking its N-terminal transmembrane domain is functional (A) Schematic representation of the Alg11 constructs. N-or C-terminal truncated constructs containing a C-terminal Protein A (ZZ)-epitope for detection were engineered as indicated into expression vector pVT100 and transformed into Δalg11. (B) Localization of Alg11 constructs. Microsomal membranes and cytosolic fraction from cells expressing the various Alg11 constructs were analyzed by Western blot. Equivalent amounts of membranes and cytosol corresponding to 10 7 cells were applied. (C) Membrane association of Alg11. Membranes from cells expressing full length Alg11 1-548 , and Alg11 lacking the N-terminal TMD, Alg11 45-548 , were subjected to treatment with 0.2% SDS, 2% NP-40 or 100 mM Na 2 CO 3 (pH 11) and further centrifuged at 100 000 g. Equivalent amounts from the pellet (P) and supernatant (S) were analyzed by Western blotting. (D) Growth of Δalg11 cells harbouring an empty vector or the different Alg11 constructs, as indicated, were incubated at 25, 34 or 36 °C. (E) Glycosylation status of CPY analyzed by Western blotting. Experimental conditions were as in Figure 1. (F) Determination of mannosyltransferase activity of the Alg11 45-548 -ZZ variant (right panel), immunoprecipitated from solubilized extract; assay II was used. As control an extract from cells expressing a non-tagged Alg11 was immunoprecipitated.

Figure 5

 5 Figure 5 Site specific mutagenesis of Alg11 (A and B) Exploring the EX 7 E motif. Mutations were introduced in the conserved EX 7 E motif of plasmid borne Alg11 ZZ as indicated and transformed into Δalg11. Their role was investigated with respect to functional complementation of growth of Δalg11 cells (A) and glycosylation of CPY monitored by Western blot (B). (C and D) Identification of K319 as an essential amino acid for Alg11 function. Various conserved lysine, glutamate and aspartate residues, with a potential role in Alg11 function were exchanged by site specific mutagenesis as indicated. Plasmid borne Alg2 was transformed into Δalg11 and tested for complementation of the growth defect of alg2 and regarding underglycosylation of CPY. (E and F) Investigation of a glycine rich motif.

Figure 6

 6 Figure 6Pathway of early lipid-linked oligosaccharide formation on the cytosolic side of the ER membrane Biosynthesis starts with the transfer of a GlcNAc-phosphate to dolichol phosphate with formation of the pyrophosphate bond, catalyzed by Alg7. The second step is catalyzed be the dimeric Alg14 / Alg13 complex, whereby membrane bound Alg14 recruits cytosolic Alg13 to the membrane with formation of the active GlcNAc transferase. Subsequent to the addition of the β1,4-linked mannose catalyzed by Alg1, bifunctional Alg2 adds both the α1,3-and α1,6linked mannose residues. The two final α1,2-mannose residues are transferred by Alg11, before the Man 5 GlcNAc 2 -PP heptasaccharide is translocated across the ER membrane to the lumen through action of an ATP-independent flippase, where further elongation occurs to the

Biochemical

  Fig.1

7 *

 7 Biochemical Journal Immediate Publication. Published on 24 Nov 2009 as manuscript BJ20091121 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091121 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Fig. 2

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  Fig. 5

Fig. 6 Biochemical

 6 Fig. 6

  Biochemical Journal Immediate Publication. Published on 24 Nov 2009 as manuscript BJ20091121 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091121

	Mutants	Motif	Complementation	Complementation	Conservation	Conservation
			of glycosylation defect of Δalg11	of growth defect of Δalg11	ALG11Yeast / ALG11Human	ALG11 Yeast/ ALG2 Yeast
	G84A	G loop	Yes	Yes	C	C
	G84P	G loop	Yes	Yes	C	C
	G85A	G loop	Yes	Yes	C	C
	G85P	G loop	No	No	C	C
	G87A	G loop	Yes	Yes	C	C
	G87P	G loop	No	No	C	C
	E88A	G loop	Yes	Yes	C	E/R
	E405A	EX 7 E	Poor	Yes	C	C
	H406A	EX 7 E	Poor	Yes	C	C
	F407A	EX 7 E	Partial	Yes	C	C
	V412A	EX 7 E	Yes	Yes	C	V/L
	E413A	EX 7 E	Yes	Yes	C	C
	E405A/ E413A	EX 7 E	No	No	C	C
	E405A/ H406A	EX 7 E	No	No	C	C
	K302A	D/E/K	Yes	Yes	K/T	C
	E318A		Yes	Yes	C	E/F
	K319A		Poor	Poor/No	C	K/E
	K322A		Yes	Yes	K/P	C
	K343A		Yes	Yes	C	C
	E356A		Yes	Yes	C	C
	K381A		Yes	Yes	K/I	K/Q
	D386A		Yes	Yes	C	D/V
	K387A		Yes	Yes	K/E	K/S
	D462A		Yes	Yes	C	C
	D476A		Yes	Yes	D/E	D/E
	D478A		Yes	Yes	D/R	D/N
	D502A		Yes	Yes	D/-	D/K
	D504A		Yes	Yes	D/-	D/I

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of Angelika Rechenmacher. We are also grateful to Dr. J. Stolz for providing plasmids and helpful discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Körber-Stiftung.

ABBREVIATIONS

ER, endoplasmic reticulum, alg, asparagine-linked glycosylation; CPY, carboxypeptidase Y; Ipp, immunoprecipitate; LLO, lipid-linked oligosaccharide; OD, optical density; OST, oligosaccharyltransferase; protein A, ZZ; TMD, transmembrane spanning domain;