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• W2015530645 abstract "In the yeast Saccharomyces cerevisiaemany of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 α-1,6-linked mannoses to which are attached many branches consisting of α-1,2-linked and α-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of theMNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesisin vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the α-1,6-polymerizing activity of the complex. In the yeast Saccharomyces cerevisiaemany of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 α-1,6-linked mannoses to which are attached many branches consisting of α-1,2-linked and α-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of theMNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesisin vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the α-1,6-polymerizing activity of the complex. The glycoproteins of the cell wall of the yeastSaccharomyces cerevisiae are modified with bothN-linked and O-linked glycans. TheO-linked structures are chains of 4–5 mannoses attached to serines and threonines, whereas the N-linked glycans comprise the conventional core structure to which can be attached a long outer chain structure of up to 200 mannose residues (1Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (439) Google Scholar, 2Orlean P. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces Cerevisiae. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 229-362Google Scholar). This “mannan” modification is so extensive that the mannoproteins contribute up to 40% of the dry weight of the yeast cell wall (3Klis F.M. Yeast. 1994; 10: 851-869Crossref PubMed Scopus (483) Google Scholar). Mannans provide an external layer to the wall, which is believed both to contribute to its structural integrity and serve to exclude hydrolytic enzymes. Analysis of the structure of the S. cerevisiae mannan shows that it consists of a long α-1,6-linked backbone of about 50 residues to which short branches of 3–4 residues in length are attached (1Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (439) Google Scholar, 4Ballou L. Hernandez L.M. Alvarado E. Ballou C.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3368-3372Crossref PubMed Scopus (60) Google Scholar, 5Peat S. Turvey J.R. Doyle D. J. Chem. Soc. 1961; : 3918-3923Crossref Scopus (34) Google Scholar). The first 2 mannoses of these branches are α-1,2-linked, and the final mannose is α-1,3-linked with some branches being additionally modified by the addition of a phosphomannose residue. A similar type of mannan structure comprising a long backbone with side branches is a feature of the cell wall of all other yeast and fungi, although the precise linkages, and even sugar residues, vary extensively even between quite closely related species (6Shibata N. Ikuta K. Imai T. Satoh Y. Satoh R. Suzuki A. Kojima C. Kobayashi H. Hisamichi K. Suzuki S. J. Biol. Chem. 1995; 270: 1113-1122Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 7Ballou L. Ballou C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2790-2794Crossref PubMed Scopus (29) Google Scholar, 8Jimenez-Barbero J. Prieto A. Gomez-Miranda B. Leal J.A. Bernabe M. Carbohydr. Res. 1995; 272: 121-128Crossref Scopus (25) Google Scholar, 9Nakajima T. Ichishima E. J. Ferment. Bioeng. 1994; 78: 472-475Crossref Scopus (12) Google Scholar, 10Gemmill T.R. Trimble R.B. J. Biol. Chem. 1996; 271: 25945-25949Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This diversity may reflect a selective pressure to evade hydrolytic enzymes or immune responses that recognize a particular combination of residues and linkages. The synthesis of the mannan structure in S. cerevisiae has been extensively investigated by both genetic and biochemical means. The mannan outer chains are only attached to the N-linked glycans of proteins destined for inclusion in the cell wall or residence in the periplasmic space. The synthesis begins in the Golgi apparatus where the first α-1,6-linked mannose residue of the mannan backbone is attached to the core by the Och1p mannosyltransferase (11Nakayama K. Nagasu T. Shimma Y. Kuromitsu J. Jigami Y. EMBO J. 1992; 11: 2511-2519Crossref PubMed Scopus (243) Google Scholar). This mannose is attached to all N-linked glycans, but then only on a subset is it extended further to form the mannan backbone. On the remaining glycans a single α-1,2-linked residue is added and then an α-1,3-linked residue is added to this, and to other points on the core structure (4Ballou L. Hernandez L.M. Alvarado E. Ballou C.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3368-3372Crossref PubMed Scopus (60) Google Scholar). The extension of the mannan backbone is performed by two recently identified complexes of proteins in the cisGolgi. The first contains the proteins Mnn9p and Van1p, and the second has been shown also to include Mnn9p as well as the proteins Anp1p, Hoc1p, and Mnn11p (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar). These two complexes possess α-1,6-transferase activity in vitro, and mutations in their components can result in either complete loss of the mannan backbone (mnn9and van1) or mannan chains with a short backbone of 10–15 residues (anp1), suggesting that the two complexes act sequentially to initiate and then extend the backbone. All of these proteins are type II membrane proteins, with a single transmembrane domain, a topology typical of Golgi glycosyltransferases (13Field M.C. Wainwright L.J. Glycobiology. 1995; 5: 463-472Crossref PubMed Scopus (98) Google Scholar, 14Colley K.J. Glycobiology. 1997; 7: 1-13Crossref PubMed Scopus (283) Google Scholar). The branches on the mannan backbone are initiated and then extended by two α-1,2-mannosyltransferases encoded by MNN2 andMNN5 (15Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These two membrane proteins are not components of the Van1p- or Anp1p-containing complexes, which make the backbone. Finally the α-1,3-linked mannose and the mannose phosphate are added by Mnn1p and Mnn6p, respectively (16Ballou C.E. Kern K.A. Raschke W.C. J. Biol. Chem. 1973; 248: 4667-4673Abstract Full Text PDF PubMed Google Scholar, 17Wiggins C.A.R. Munro S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7945-7950Crossref PubMed Scopus (316) Google Scholar, 18Wang X.H. Nakayama K. Shimma Y. Tanaka A. Jigami Y. J. Biol. Chem. 1997; 272: 18117-18124Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Studies on mannan synthesis in S. cerevisiae are facilitated by mannan not being essential for viability. Survival without mannan is, however, dependent on cells being able to sense the cell wall defect, presumably to allow up-regulation of other cell wall components especially chitin (19Dallies N. Francois J. Paquet V. Yeast. 1998; 14: 1297-1306Crossref PubMed Scopus (198) Google Scholar). Thus mutations that result in defective mannans show synthetic lethality with components of the protein kinase C pathway, which regulates the expression of cell wall components (15Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,20Neiman A.M. Mhaiskar V. Manus V. Galibert F. Dean N. Genetics. 1997; 145: 637-645Crossref PubMed Google Scholar, 21Nakamura T. Ohmoto T. Hirata D. Tsuchiya E. Miyakawa T. Mol. Gen. Genet. 1997; 256: 481-487Crossref PubMed Scopus (9) Google Scholar). Cell wall integrity is likely to be particularly critical during budding, and again survival without mannan seems also to depend both on the actin cytoskeleton being well organized and on a functional mitotic check point, which allows bud formation to be slowed (22Mondesert G. Reed S.I. J. Cell Biol. 1996; 132: 137-151Crossref PubMed Scopus (34) Google Scholar, 23Mondesert G. Clarke D.J. Reed S.I. Genetics. 1997; 147: 421-434Crossref PubMed Google Scholar, 24Sekiya-Kawasaki M. Botstein D. Ohya Y. Genetics. 1998; 150: 43-58PubMed Google Scholar). In this study, we investigate a Golgi membrane protein of previously unknown function that was initially identified as corresponding to a bud emergence delay mutation bed1 (22Mondesert G. Reed S.I. J. Cell Biol. 1996; 132: 137-151Crossref PubMed Scopus (34) Google Scholar). It was subsequently shown to correspond also to a previously identified mannan synthesis mutant mnn10 (25Ballou L. Cohen R.E. Ballou C.E. J. Biol. Chem. 1980; 255: 5986-5991Abstract Full Text PDF PubMed Google Scholar, 26Dean N. Poster J.B. Glycobiology. 1996; 6: 73-81Crossref PubMed Scopus (34) Google Scholar). Because Mnn10p is distantly related to Mnn11p, one of the components of the Anp1p-containing complex, we investigated its relationship to this complex. We show here that it is a further component of the complex that seems to comprise five proteins. This large structure does not appear to contain further proteins. Investigation of the activity of the complex in wild type and mutant strains indicates that the Mnn10p and Mnn11p proteins are responsible for the majority of the α-1,6-polymerizing activity of the complex. S. cerevisiae strain SEY6210 (Matα ura3–52 his3Δ200 leu2–3,112 trp-Δ901 lys2–801 suc2-Δ9) and derivatives were used throughout (27Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (732) Google Scholar). Genes were disrupted by replacement of the open reading frame, or tagged at the exact C terminus using homologous recombination with polymerase chain reaction products containing Schizosaccharomyces pombe HIS5 orKluyveromyces lactis URA3 genes, and integrants checked with polymerase chain reaction (28Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1113) Google Scholar, 29Wach A. Brachat A. AlbertiSegui C. Rebischung C. Philippsen P. Yeast. 1997; 13: 1065-1075Crossref PubMed Scopus (507) Google Scholar). For protein A fusions cleavable by the tobacco etch virus (TEV) 1The abbreviations used are: TEV, tobacco etch virus; HA, hemagglutinin; Endo H, endoglycosidase H; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; M-Pol, mannan polymerase.protease, plasmid pZZ-HIS5 was used as described previously (15Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). For hemagglutinin (HA) epitope tagging, polymerase chain reaction products were amplified from plasmid p3xHA-HIS5 that encodes a linker of amino acid sequence GAGAGA, followed by three copies of the sequence YPYDVPDYA, with the first and third repeats followed by a G residue. For Myc-tagging of open reading frames, a similar plasmid with nine copies of the Myc tag was used 2I. Adams and J. V. Kilmartin, unpublished material. except for Myc-tagged invertase, which was expressed in strains from a constitutive promoter using an integration plasmid pTi Invmyc (26Dean N. Poster J.B. Glycobiology. 1996; 6: 73-81Crossref PubMed Scopus (34) Google Scholar). Proteins separated by SDS-polyacrylamide gel electrophoresis were electrophoresed onto nitrocellulose and probed with antibodies in phosphate-buffered saline, 2% dried milk, 0.5% Tween 20. Immunoblotting and immunoprecipitations were performed with monoclonals against the Myc epitope (9E10), the HA epitope (12CA5), or ribosomal protein Tcm1p (kindly provided by Jon Warner) or with rabbit antisera against both tags (Santa Cruz Biotechnology) or Anp1p (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar). Peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech). Total protein samples were prepared by resuspending log-phase yeast at oneA 600 unit per 20 μl of SDS buffer, bead beating for 5 min at 4 °C (425–600-μm glass beads, Sigma), and denaturing at 65 °C for 5 min. Immunoprecipitates were prepared from unlabeled cells as described previously (15Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Precipitation of protein A fusions was performed essentially as described previously but adapted for metabolically labeled cells. Thus four A 600units of log-phase cells were labeled with 200 μCi of Tran35S-label (1,170 Ci/mmol, ICN) in 0.5 ml for 30 min at 30 °C, spheroplasted as for the unlabeled cells, and resuspended in 0.5 ml of T/D/I buffer (10 mm triethanolamine, pH 7.5, 150 mm NaCl, 1% digitonin, 2 mm EDTA supplemented by protease inhibitors). After centrifugation at 14,000 ×g for 10 min to remove cell debris, the supernatant was added to 15 μl of IgG-Sepharose (Amersham Pharmacia Biotech), rocked gently overnight, and the beads washed five times for 1 h in 0.4 ml T/D/I, before cleavage overnight in 20 μl of T/D/I with 5 units of TEV protease (Life Technologies, Inc.), all steps after spheroplasting being at 4 °C. The supernatant from the beads was removed, divided in two and treated with, or without, 500 units of endoglycosidase H (Endo H) according to the manufacturer's instructions (New England Biolabs). Protein was precipitated by methanol/chloroform extraction, resuspended in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and the gel was treated with Amplify (Amersham Pharmacia Biotech) and fluorographed. Protein complexes prepared by IgG-Sepharose precipitation and TEV protease cleavage from 1,000A 600 units of cells, or 14,000 ×g supernatants from spheroplasts lysed in T/D/I buffer, were fractionated on a 34 × 1.5-cm column of Sephacryl S400 (Amersham Pharmacia Biotech). The column was run in 10 mmtriethanolamine, pH 7.5, 150 mm NaCl, 1 mmEDTA, 0.4% digitonin at 15 ml/h and 1 ml of fractions were collected. 100 μl of fractions were precipitated by methanol/chloroform extraction with soybean trypsin inhibitor as a carrier, resuspended in SDS buffer, and analyzed by immunoblotting. For standards, thyroglobulin, catalase, and ferritin (Amersham Pharmacia Biotech) were run on the same column under identical conditions and analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. Protein A fusions or Anp1p-containing complexes were isolated and used on the beads for mannosyltransferase reactions as described previously (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar, 15Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The reaction products were fractionated by gel filtration on a 14 × 1.0-cm Sephadex G10 column (Amersham Pharmacia Biotech) run in deionized water. The radioactive peak fractions were pooled, concentrated by lyophilizing, treated with mannosidases, and the products reacted with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) for separation by electrophoresis as described previously (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar, 30Jackson P. Methods Enzymol. 1994; 230: 250-265Crossref PubMed Scopus (105) Google Scholar) with the reaction products from 50 to 100 A 600 units of cells loaded per lane. The resulting gels were exposed to a PhosphorImager screen for 3 days (or 2 weeks for theΔmnn11 and Δmnn10 experiments) and analyzed with a Molecular Dynamics PhosphorImager and ImageQuant software. To determine whether Mnn10p was a component of one of the two Mnn9p-containing complexes, the same co-immunoprecipitation approach was followed that was originally used to reveal the interactions between Mnn9p and the other members of the complexes (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar). Thus a triple HA-epitope tag was inserted at the end of theMNN10 open reading frame by homologous recombination. Protein blotting revealed that the tagged protein migrated as a single band with an apparent molecular mass of 46 kDa, reasonably close to that predicted from the sequence of the protein and the epitope tag (49 kDa, Fig. 1 A). An anti-HA monoclonal antibody was used to precipitate the protein from yeast solubilized with the mild detergent digitonin. The immunoprecipitates were then blotted with antibodies to Anp1p and Van1p, and Fig.1 B shows that Mnn10p is associated with Anp1p and not with Van1p. Moreover, blotting of the supernatant following precipitation revealed that the Anp1p was quantitatively precipitated by the tagged Mnn10p, indicating that all of the Anp1p is associated with complexes that contain Mnn10p (Fig. 1 B). Our previous analysis of the Mnn9p-containing complexes had shown that Anp1p is associated not only with Mnn9p but also with two other membrane proteins, Hoc1p and Mnn11p. As with Mnn10p, these proteins were not found associated with the Van1p-Mnn9p complex. This raises the question of how these five proteins are organized. In each case it is known that the protein associates with Anp1p. One possibility is that all five proteins are in a single complex. Alternatively there could be distinct complexes containing Mnn9p-Anp1p and one or two of the other components. We addressed this issue in two ways. First, we constructed strains with different epitope tags at the C terminus of pairs of the proteins. Immunoprecipitates were then performed using one anti-tag antibody, and the precipitated proteins probed on a blot for the second tagged protein. A representative experiment is shown in Fig.2 A for strains expressing Mnn10p and Mnn11p, tagged with Myc and HA in both combinations. In both cases, not only could the HA-tagged member of the pair be precipitated with the anti-HA monoclonal antibody as expected, but it could also be precipitated with the anti-Myc monoclonal antibody. This indicates that Mnn10p and Mnn11p are present in the same complex. Likewise theHOC1 open reading frame was tagged with the Myc epitope in strains in which either Mnn10p or Mnn11p were tagged with HA. In both cases the Myc-tagged Hoc1p could be precipitated with antibodies to the Myc or the HA tags, and also with a rabbit polyclonal against Anp1p. Blotting of the digitonin lysate of the Hoc1p-Myc-expressing cells revealed two bands, of which only the upper is present in blots of cellular proteins solubilized directly in SDS-containing sample buffer (Fig. 2 B and data not shown). Because the tag is at the C terminus, this suggests that proteases in the digitonin lysate remove about 5 kDa from the NH2 terminus of the protein. Such clipping in the region after the amino-terminal transmembrane domain has been seen for many Golgi enzymes in both yeast and mammals and is proposed to reflect this part of the enzymes being a flexible stalk region (31Häusler A. Robbins W. Glycobiology. 1992; 2: 77-84Crossref PubMed Scopus (62) Google Scholar, 32Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar). Interestingly, this clipped form does not co-precipitate with either Mnn10p, Mnn11p or Anp1p, which may reflect removal by the protease of a region required for association in the complex (Fig. 2 B). The only exception to this co-precipitation of the various Anp1p-associated proteins was observed when tagged Hoc1p was precipitated from a strain also expressing tagged Mnn10p. Although precipitation of HA-tagged Mnn10p with anti-HA resulted in the co-precipitation of Myc-tagged Hoc1p, only low levels of Mnn10p-HA were precipitated when the precipitation was performed the other way round with anti-Myc (Fig. 2 B). One possibility is that the C-terminal tag on Hoc1p lies close to the interface between Hoc1p and Mnn10p, and that binding of the anti-Myc monoclonal antibody disrupts the interaction. To investigate further the composition of the Anp1p-containing complex and examine the possibility that it contains yet further components, a second approach was used to address the composition of the complex. For this a protein A tag was attached to the C terminus of each of the components of the complex in separate strains. The spacer between the protein and the tag contained a cleavage site for the sequence-specific protease from TEV. This allows the tagged proteins to be captured on IgG-Sepharose, and then proteolytically eluted under native conditions with minimal background (15Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The isolation was initially performed with strains expressing either protein A-tagged Anp1p or tagged Van1p, or no tagged protein. Fig. 3 A shows that the two tagged proteins precipitate a distinct pattern of bands, consistent with their being in two distinct complexes that share only the component Mnn9p. This analysis was then extended to the other components of the Anp1p-containing complex, and Fig. 3 Bshows the results of such a precipitation and elution performed on35S-labeled strains in which the different components of the complex had been tagged. A thirteen residue spacer including the TEV cleavage site is left on the protein following protease treatment, and this results in a small reduction of mobility compared with the nontagged version. In most cases all the other proteins can be identified as co-precipitating with a particular component. Analysis of the gels is complicated by the fact that Mnn10p and Hoc1p apparently co-migrate (consistent with their predicted molecular weights differing by only 0.4 kDa). However, when Mnn10p is shifted following the addition of the tag, a band the size of Hoc1p remains. Interestingly in the converse case, when the tag is attached to Hoc1p, only a very faint band in the position of Mnn10p is observed. This is consistent with the immunoprecipitation results described above, and with the notion that the C terminus of Hoc1p may be close to the interface with Mnn10p. Taken together, these results strongly suggest that there are two different Mnn9p-containing complexes in the yeast cis Golgi. The first comprises Van1p and Mnn9p, and the second Anp1p, Mnn9p, Hoc1p, Mnn10p, and Mnn11p. Careful examination of the autoradiograms did not reveal any further unidentified bands in the Anp1p complex suggesting that these five proteins are the major constituents of the complex. However it remains possible that there are further proteins present at stoichiometry too low for detection by this approach. We have previously shown that both these complexes have α-1,6-mannosyltransferase activity in vitro, attaching multiple residues to make polymeric structures. To simplify further discussion, we propose to refer to these two complexes as mannan polymerases (M-Pol) I and II, respectively. The above analysis indicates that M-Pol II contains three more components than M-Pol I. As such it would be expected to be a larger structure, and this prediction was tested using gel filtration. The protease elution method described above allows the complexes to be released from the beads in an assembled state, and such released complexes were applied to a gel filtration column. Fig.4 A shows that the two complexes do indeed elute from such a column with very different profiles. The Van1p complex (M-Pol I) elutes with a predicted size of about 280 kDa, whereas the Anp1p complex (M-Pol II) is apparently much larger with a size in excess of 1,000 kDa. The large size of M-Pol II is unlikely to be a result of the precipitation and cleavage protocol used to prepare it. When a whole cell lysate was applied to the same column, Anp1p elutes in a similar position (Fig.4 B) and an epitope-tagged version of Mnn9p elutes in two peaks corresponding to the positions of the isolated M-Pol I and M-Pol II. The individual predicted molecular masses of Van1p and Mnn9p total 107 kDa (plus the N-linked glycan on Van1p, which adds about 5 kDa to its apparent molecular weight on an SDS-polyacrylamide gel), whereas those of the components of M-Pol II total 245 kDa. To maintain the solubility of these membrane proteins the column was run in the presence of digitonin, which forms micelles of 70 kDa in 0.1m NaCl (33Smith E.L. Pickels E.G. Proc. Natl. Acad. Sci. U. S. A. 1940; 26: 23658-23663Crossref Google Scholar). Although this sort of gel filtration analysis cannot be expected to give very accurate sizes, it seems likely that at least some of the proteins are present in the M-pol complexes at a greater than monomeric stoichiometry. It has been previously shown that M-Pol I and M-Pol II have both α-1,6- and α-1,2-mannosyltransferase activity in vitro, raising the question of what contribution the individual components of the complex make to this activity (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar). To address this issue, we initially examined the effect of deleting individual subunits on mannan synthesis in vivo. It is known that mutations in Mnn10p result in reduction in the length of mannan chains, but the effect of loss of Mnn11p has not previously been reported. The protein invertase is modified with mannan chains on 8–10 of its N-linked glycans, and Fig.5 A shows that the gel mobility of invertase is increased in yeast in which either Anp1p, Mnn10p or Mnn11p is absent. Consistent with this the Δmnn11 strain also shows increased sensitivity to hygromycin, a property of other mannan defective mutants including anp1 and mnn10(data not shown). This demonstrates that Mnn11p is required for the synthesis of full-length mannan chains in vivo, and justifies our inclusion of the gene in the MNN class as defined by Ballou and co-workers (34Raschke W.C. Kern K.A. Antalis C. Ballou C.E. J. Biol. Chem. 1973; 248: 4660-4666Abstract Full Text PDF PubMed Google Scholar, 35Ballou C.E. Methods Enzymol. 1990; 185: 440-470Crossref PubMed Scopus (273) Google Scholar). It has been observed previously that removal of the Mnn9p component of M-Pol II results in the destabilization of Anp1p (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar, 36Hashimoto H. Yoda K. Biochem. Biophys. Res. Commun. 1997; 241: 682-686Crossref PubMed Scopus (36) Google Scholar). This presumably reflects a requirement for association between the two proteins for correct folding. In contrast, deletion of the other members of the complex does not result in the complete loss of Anp1p (Fig. 5 B). However, the levels are reduced in some cases, and normalization to a control ribosomal protein (Tcm1p) shows that in the Δmnn10 and Δmnn11 strains the level of Anp1p is reduced by 85 and 75%, respectively, whereas deletion ofHOC1 has no effect. This suggests that Mnn10p and Mnn11p contribute to the folding or stability of M-Pol II. Because the level of Anp1p is reduced in the Δmnn10 and Δmnn11strains, it is possible that the defect in mannan synthesis seen is not because the proteins are directly involved in mannan synthesis, but rather because they are required for the stability or integrity of other proteins in the complex, which are actually responsible for mannan synthesis. To address the catalytic role of Mnn10p and Mnn11p, the Anp1p-protein A fusion described above was used to isolate M-Pol II for examination of its enzymatic activity in vitro. We initially examined the activity of the M-Pol II from wild type cells, and compared it with that of M-Pol I. Thus Anp1p and Van1p protein-A fusions were precipitated from cells and incubated on the beads with labeled GDP mannose and α-1,6-mannobiose as an acceptor. The products of the transferase reactions were divided into aliquots and digested with combinations of mannosidases. Finally, the charged fluorophore ANTS was attached to the reducing ends of the products to allow analysis by fluorophore-assisted carbohydrate electrophoresis (30Jackson P. Methods Enzymol. 1994; 230: 250-265Crossref PubMed Scopus (105) Google Scholar). The gels were autoradiographed and examined by fluorescence to examine the reaction products. Fig. 6 shows that both M-Pol I and M-Pol II produced a ladder of mannosylated products as previously observed (12Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (185) Google Scholar). Mannosidase digestion revealed that the product of M-Pol II is almost completely digested to monomeric mannose by α-1,6-mannosidase, suggesting that this is the major linkage made by the complex. In contrast," @default.
• W2015530645 created "2016-06-24" @default.
• W2015530645 creator A5054717950 @default.
• W2015530645 creator A5059008048 @default.
• W2015530645 creator A5062767154 @default.
• W2015530645 date "1999-03-01" @default.
• W2015530645 modified "2023-10-16" @default.
• W2015530645 title "The Saccharomyces cerevisiae Protein Mnn10p/Bed1p Is a Subunit of a Golgi Mannosyltransferase Complex" @default.
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