FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2138242591> ?p ?o ?g. }

• W2138242591 endingPage "36262" @default.
• W2138242591 startingPage "36254" @default.
• W2138242591 abstract "N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc2-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc2-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor. N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc2-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc2-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor. Asparagine (N)-glycosylation is an essential modification that regulates protein folding and stability. Prior to its attachment to protein, the oligosaccharide Glu3Man9GlcNAc2 is assembled on the lipid carrier, dolichyl pyrophosphate (dol-PP), in the ER 2The abbreviations used are:ERendoplasmic reticulumLLOlipid-linked oligosaccharideALGasparagine-linked glycosylationdoldolicholORFopen reading frameHAhemagglutininCPYcarboxypeptidase YGFPgreen fluorescent proteinPPpyrophosphate (see Refs. 1Burda P. Aebi M. Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar, 2Abeijon C. Hirschberg C.B. Trends Biochem. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 3Kornfeld R. Kornfeld S. Ann. Rev. of Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar, 4Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (440) Google Scholar for review). The earliest steps of this lipid-linked oligosaccharide (LLO) synthesis begin on the cytoplasmic face of the ER. Seven sugars, (two N-acetylglucosamines and five mannoses) are sequentially added to dol-P to form Man5GlcNAc2-PP-dol by enzymes that have their catalytic domain on the cytosolic side of the ER membrane and use sugar nucleotide substrates (2Abeijon C. Hirschberg C.B. Trends Biochem. Sci. 1992; 17: 32-36Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 5Snider M.D. Rogers O.C. Cell. 1984; 36: 753-761Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 6Perez M. Hirschberg C.B. J. Biol. Chem. 1986; 261: 6822-6830Abstract Full Text PDF PubMed Google Scholar). The enzymes that catalyze addition of the next seven sugars (four mannoses and three glucoses) do so within the lumen of the ER and use dolichol-linked sugar substrates (see Ref. 1Burda P. Aebi M. Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (528) Google Scholar for review). Once assembled, this core oligosaccharide is transferred to protein by oligosaccharyltransferase through an N-glycosidic bond to an asparagine that is part of the Asn-X-(Ser/Thr) consensus sequence (7Yan A. Lennarz W.J. J. Biol. Chem. 2005; 280: 3121-3124Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Protein-linked oligosaccharide is immediately modified by the removal of glucoses and mannose by ER glucosidases and mannosidases. Failure to properly synthesize, transfer, or modify the N-linked glycan results in glycoproteins that are recognized by the quality control systems that restrict these aberrant proteins from exiting the ER to the Golgi and target their degradation (for review see Refs. 8Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1620) Google Scholar and 9Hebert D.N. Garman S.C. Molinari M. Trends Cell Biol. 2005; 15: 364-370Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). endoplasmic reticulum lipid-linked oligosaccharide asparagine-linked glycosylation dolichol open reading frame hemagglutinin carboxypeptidase Y green fluorescent protein pyrophosphate In the yeast, Saccharomyces cerevisiae, many of the glycosyltransferases that catalyze synthesis of the core oligosaccharide have been identified. All of the ALG genes (asparagine-linked glycosylation) identified thus far (ALG1-12) encode membrane-spanning glycosyltransferases that have their catalytic domains on one or the other side of the ER membrane, consistent with the topological constraints of this pathway (10Couto J.R. Huffaker T.C. Robbins P.W. J. Biol. Chem. 1984; 259: 378-382Abstract Full Text PDF PubMed Google Scholar, 11Jackson B.J. Kukuruzinska M.A. Robbins P.W. Glycobiology. 1993; 3: 357-364Crossref PubMed Scopus (55) Google Scholar, 12Aebi M. Gassenhuber J. Domdey H. te Heesen S. Glycobiology. 1996; 6: 439-444Crossref PubMed Scopus (115) Google Scholar, 13Reiss G. te Heesen S. Zimmerman J. Robbins P.W. Aebi M. Glycobiology. 1996; 6: 493-498Crossref PubMed Scopus (85) Google Scholar, 14Barnes G. Hansen W.J. Holcomb C.L. Rine J. Mol. Cell. Biol. 1984; 4: 2381-2388Crossref PubMed Scopus (78) Google Scholar, 15Stagljar I. te Heesen S. Aebi M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5977-5981Crossref PubMed Scopus (93) Google Scholar, 16Burda P. te Heesen S. Brachat A. Wach A. Dusterhoft A. Aebi M. Proc. Natl. Acad. Sci. 1996; 93: 7160-7165Crossref PubMed Scopus (91) Google Scholar, 17Frank C.G. Aebi M. Glycobiology. 2005; (in press)Google Scholar, 18Burda P. Aebi M. Glycobiology. 1998; 8: 455-462Crossref PubMed Scopus (94) Google Scholar, 19Cipollo J.F. Trimble R.B. Chi J.H. Yan Q. Dean N. J. Biol. Chem. 2001; 276: 21828-21840Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Burda P. Jakob C.A. Beinhauer J. Hegemann J.H. Aebi M. Glycobiology. 1999; 9: 617-625Crossref PubMed Scopus (80) Google Scholar). In all organisms studied thus far, ALG mutations affecting the cytosolic reactions leading to Man5GlcNAc2-PP-dol synthesis result in lethality or very severe phenotypes (19Cipollo J.F. Trimble R.B. Chi J.H. Yan Q. Dean N. J. Biol. Chem. 2001; 276: 21828-21840Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Huffaker T.C. Robbins P.W. J. Biol. Chem. 1982; 257: 3203-3210Abstract Full Text PDF PubMed Google Scholar, 22Huffaker T.C. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar). Mutations affecting the later, luminal intermediates of LLO synthesis manifest as a range of phenotypes, from undetectable in S. cerevisiae (12Aebi M. Gassenhuber J. Domdey H. te Heesen S. Glycobiology. 1996; 6: 439-444Crossref PubMed Scopus (115) Google Scholar, 13Reiss G. te Heesen S. Zimmerman J. Robbins P.W. Aebi M. Glycobiology. 1996; 6: 493-498Crossref PubMed Scopus (85) Google Scholar, 15Stagljar I. te Heesen S. Aebi M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5977-5981Crossref PubMed Scopus (93) Google Scholar, 16Burda P. te Heesen S. Brachat A. Wach A. Dusterhoft A. Aebi M. Proc. Natl. Acad. Sci. 1996; 93: 7160-7165Crossref PubMed Scopus (91) Google Scholar, 18Burda P. Aebi M. Glycobiology. 1998; 8: 455-462Crossref PubMed Scopus (94) Google Scholar, 22Huffaker T.C. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar, 23te Heesen S. Lehle L. Weissmann A. Aebi M. Eur. J. Biochem. 1994; 224: 71-79Crossref PubMed Scopus (96) Google Scholar) to the heterogenous phenotypes associated with congenital disorders of glycosylation (CDG) in humans (24Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 25Freeze H.H. Biochim. Biophys. Acta. 2002; 1573: 388-393Crossref PubMed Scopus (67) Google Scholar). All of the ALG genes are highly conserved among eukaryotes, although certain unicellular eukaryotes are missing a subset (26Samuelson J. Banerjee S. Magnelli P. Cui J. Kelleher D.J. Gilmore R. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1548-1553Crossref PubMed Scopus (215) Google Scholar). These unicellular organisms consequently produce lipid-linked oligosaccharides that are shorter than Glu3Man9GlcNAc2PP-dol that is produced in S. cerevisiae and in humans. With the exception of Giardia lamblia and Plasmodium falciparum, which produce LLO lacking mannose and glucose, the shortest glycan produced by all eukaryotes that have been examined is Man5GlcNAc2-PP-dol (26Samuelson J. Banerjee S. Magnelli P. Cui J. Kelleher D.J. Gilmore R. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1548-1553Crossref PubMed Scopus (215) Google Scholar). The evolutionary conservation and essentiality of the proteins synthesizing Man5GlcNAc2-PP-dol underscore the importance of this glycan and the reactions that occur on the cytosolic face of the ER that lead to its assembly. Despite its importance, the identity of several of the enzymes that catalyze the reactions on the cytosolic face of the ER remains a mystery. Using a bioinformatics approach, two S. cerevisiae ORFs (YGL047w and YBR070c) were recently identified that are distantly related to the bacterial MurG UDP-GlcNAc glycosyltransferase involved in peptidoglycan synthesis and that are implicated in playing a role in N-linked glycosylation (27Chantret I. Dancourt J. Barbat A. Moore S.E. J. Biol. Chem. 2005; 280: 9236-9242Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Attenuated expression of either of these genes affects the second step of lipid-linked oligosaccharide synthesis, the addition of a β1,4-linked GlcNAc to GlcNAc-PP-dol that produces GlcNAc2-PP-dol on the cytosolic face of the ER (27Chantret I. Dancourt J. Barbat A. Moore S.E. J. Biol. Chem. 2005; 280: 9236-9242Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Remarkably, unlike any other eukaryotic ER glycosyltransferases, Ygl047w, which we designate here as Alg13, is predicted to contain a consensus glycosyltransferase catalytic domain but lacks any predicted membrane-spanning domains. The other polypeptide, Ybr070c, which we designate as Alg14, contains several predicted membrane-spanning domains but lacks any sequences that are predicted to participate in sugar transfer. Because Alg13 contains a domain that is strongly related to the catalytic domain found in other UDP-sugar transferases and displays a mutant phenotype that demonstrates its involvement in LLO synthesis, it seems likely to play a direct role in UDP-GlcNAc transfer. The role of Alg14 is less clear, particularly because “split” glycosyltransferases are unprecedented among eukaryotes. In this work we have tested the hypothesis that Alg13 and Alg14 interact on the ER face to form a functional hetero-oligomeric UDP-GlcNAc glycosyltransferase whose membrane-spanning domain and catalytic domain are on separate polypeptides. We demonstrate that these enzymes physically interact with one another on the ER membranes, and that Alg14 is required to recruit Alg13 from the cytosol to the ER. Furthermore although neither of the human Alg13 and Alg14 orthologues can interact with their yeast partners, when co-expressed these proteins can functionally complement the loss of ALG13 or ALG14. We speculate that this enzyme has evaded identification because it is an unusual hetero-oligomeric glycosyltransferase, comprised of at least two distinct polypeptides, both of which are required for LLO biosynthesis. Yeast Strains and Media—Yeast strains used in this study are listed in TABLE ONE. W303a (MATa ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100) is the parental strain for all the strains used in work. Epitope tagging and replacement of promoters or chromosomal loci employed PCR-mediated recombination (28Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1120) Google Scholar) using the standard pFA6a-series of plasmids as template (29Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4171) Google Scholar). The pFA6a-3FLAG-His3MX6, a derivative of pFA6a-3HA-His3MX6 was kindly provided by M. Umemura. Standard yeast media, growth conditions, and genetic techniques were used (30Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 3-20Crossref PubMed Scopus (2543) Google Scholar).TABLE ONEStrains used in this studyStrainGenotypeRef.W303aMATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3, 112 can1-100(37)XGY31As in W303a and Gallpr-ALG1::kanmX4(32)XGY151As in W303a and Gallpr-3HA-ALG14::his5+This studyXGY154As in W303a and Gallpr-3HA-ALG13::his5+ trp1::RPT6-TRP1This studyXGY155As in W303a and ALG13-3FLAG::his5+This studySKY202As in W303a and ALG13-3HA::his5+This studySKY203As in W303a and ALG13-FLAG::URA3 Gallpr-3HA-ALG14::his5+This studyXGY156As in W303a and ALG13-3FLAG::his5+ GAL1pr-3HA-ALG14-TRP1This studyXGY158As in W303a and ALG13-3FLAG::his5+ GAL1pr-3HA-ALG14-TRP1 ura3::ALG14pr-3HA-ALG14-URA3This studyXGY166As in W303a and ADE2 ura3::Kar2-mRFP-HDEL-URA3This studyXGY167AAs in W303a and ADE2 ALG13-GFP-his5+ ura3::Kar2-mRFP-HDEL-URA3This studyXGY168As in W303a and ADE2 ALG14-GFP-his5+ ura3::Kar2-mRFP-HDEL-URA3This study Open table in a new tab In XGY151 and XGY154, the ALG14 and ALG13 chromosomal promoter sequences including the initiating ATG (220 and 202 base pairs upstream the initiating ATG, respectively) were replaced by the GAL1 promoter followed by a start codon and the triple HA epitope marked with the Schizosaccharomyces pombe his5+gene. XGY154 also contain an additional copy of the RPT6 gene and its upstream promoter sequence integrated at the trp1 locus. RPT6 is an essential gene that lies adjacent to ALG13 but that is transcribed from the opposite strand of chromosome VII. We found that replacement of the ALG13 promoter with the GAL1 promoter interferes with normal RPT6 expression. Therefore a DNA fragment, containing the RPT6 ORF and 267 bp of 5′-flanking sequence, was cloned into the TRP1 integration vector, pRS304 (31Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), linearized, and integrated at the trp1 locus in XGY154. SKY202 was created by replacing the ALG13 allele with a C-terminal HA-tagged ALG13 allele, marked with the S. pombe his5+ gene. The XGY155 strain contains a replacement of the chromosomal ALG13 locus with a C-terminal triple FLAG-tagged ALG13 allele, marked by the S. pombe his5+ gene. XGY156 was generated from XGY155 by replacing 220 base pairs upstream of the ALG14 chromosomal locus with the GAL1 promoter followed by the triple HA epitope, marked with the TRP1 gene. XGY158 was constructed from XGY156 and contains a 3HA-tagged ALG14 allele under its own promoter integrated at ura3 (by integration of pXG202 (see “Plasmid Construction” below) and in addition contains a 3HA-tagged ALG14 allele driven by the glucose repressible GAL1 promoter. XGY166 contains a normal ADE2 allele integrated at the ade2 locus, and the pTiKmRFP plasmid at ura3 locus. pTiKmRFP expresses Kar2-mRFP-HDEL (see “Plasmid Construction”, below). This strain allowed the fluorescent analysis of the Kar2-mRFP-HDEL ER marker using monomeric RFP, without interference of the red pigment that accumulates due to the ade2 mutation. Strains XGY167A and 168 were derived from XGY166 and contain a replacement of the chromosomal ALG13 and ALG14 loci, respectively, with GFP-tagged alleles that are marked with the S. pombe his5+ gene. Plasmid Constructions—Standard molecular biology techniques were used for all plasmid constructions. The correct sequence of all PCR-amplified products was verified by DNA sequencing. The sequences of primers used in this study are available upon request. To express plasmid borne genes from the ALG13 promoter, the promoter sequence of the ALG13 gene was amplified directly from the yeast genome by PCR, digested with XhoI and EcoRI and cloned into the URA3, integrative plasmid, pRS306 (31Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), to generate pXG200. The same strategy was used to create pXG201, which contains the ALG14 promoter. This PCR product was cloned into the KpnI/HindIII sites of pRS306. To express the 3HA-ALG14 gene from the ALG14 promoter, the 3HA-ALG14 gene was amplified from strain XGY151 and cloned into the HindIII/SmaI sites of pXG201 to create pXG202. Linearization of this plasmid with EcoRV within the URA3 gene targets integration at the ura3 locus. pTiKmRFP contains a KAR2-mRFP-HDEL fusion gene (with a ClaI site at the KAR2/mRF-HDEL junction) whose expression is controlled by the constitutive triose-phosphate isomerase (TPI1) promoter. This plasmid contains the 5′-region of the KAR2 ORF, which encodes the first 45 amino acids, in-frame with the mRFP (monomeric red fluorescent protein) gene that lacks its initiating ATG and that contains an HDEL-encoding sequence at the 3′-end, in the SacI/XbaI sites of pTi-Sac. Human ALG13 (GLT28D1) and ALG14 (MGC19780) cDNAs were amplified by PCR using Marathon-Ready™ cDNA derived from human whole brain (Clontech, Palo Alto, CA). The amplified fragments were digested with HinDIII and XbaI and inserted into expression vector, pFLAG-CMV4 (Sigma) to generate pXG203 (containing FLAG-hALG13) and pXG204 (containing FLAG-hALG14). To express the human ALG13-FLAG-tagged gene from the yeast ALG13 promoter, pXG203 was used as template for PCR to amplify ALG13 with a primer set encoding the FLAG peptide sequence. The product was digested with EcoRI and EcoRV, and cloned into EcoRI/SmaI sites of pXG200. This generated pXG205, a URA3 integration plasmid that expresses FLAG-tagged human ALG13 from the yeast ALG13 promoter. An XhoI/XbaI fragment including the ALG13 promoter and entire hALG13-FLAG fusion gene from pXG205 was cloned into the SalI/XbaI sites of the LEU2, integrative plasmid, pRS305, to create a similar plasmid, pXG206, but marked with LEU2. Linearization of this plasmid with EcoRV, within the LEU2 gene, targets integration at the leu2 locus. To express the human ALG14 homologue from the GAP1 promoter, pXG204 was used as a template to amplify the ALG14-FLAG ORF, with primers tailed with sequences encoding the FLAG tag. The product was digested with SacI and EcoRV, and cloned into SacI/SmaI sites of pTi-Sac (32Gao X.D. Nishikawa A. Dean N. Glycobiology. 2004; 14: 559-570Crossref PubMed Scopus (57) Google Scholar) to generate pXG207. To create pRS304RPT6, the RPT6 gene including its promoter sequence was amplified by PCR, digested with XhoI and EcoRV, and cloned into XhoI/SmaI sites of the TRP1, integrative plasmid pRS304. Subcellular Fractionation and Western Immunoblotting Analysis—Yeast were grown and harvested at logarithmic growth phase (A600 1-3). About 2 × 109 cells were incubated in 1 ml of A-Buffer (100 mm EDTA, 0.5% β-mercaptoethanol, 10 mm Tris-HCl, pH 7.5) for 15 min at 30 °C, spun down, and then resuspended in 0.8 ml of S-Buffer (1.0 m sorbitol, 2 mm MgCl2, 0.14% β-mercaptoethanol, 50 mm Tris-HCl, pH 7.5) with 50 units/A600 zymolyase 100T (SEIKAGAKU Co.). After incubating at 30 °C for 45∼60 min, during which time 70% of cells were converted to spheroplasts, cells were collected and washed twice with ice-cold S-Buffer. The cells were suspended in 1 ml of lysis buffer (0.2 m sorbitol, 1 mm EDTA, 50 mm Tris-HCl, pH 7.5) containing protease inhibitors and lysed by Dounce homogenization, with 20 strokes of a Teflon pestle on ice. Unbroken cells and debris were pelleted by centrifugation at 1000 × g for 10 min to yield a crude extract. The post 1000 × g supernatant was collected and centrifuged at 13,000 × g for 15 min, which yielded the P13 pellet and the S13 supernatant. The S13 fraction was centrifuged at 100,000 × g for 60 min to yield the P100 and S100 supernatant. Together with crude extract and S100 fraction, P13 and P100 fraction were resuspended in a suitable mount of lysis buffer and protein concentrations were determined by BCA protein assay (Pierce). 10 μg of each fraction were separated by 10% SDS-PAGE and transferred to Immobilon-PVDF membranes (Millipore). To make the membrane fraction that was used in A and B of Fig. 4, exponentially growing yeast cells (10 A600) were homogenized by vortexing with glass beads in ice-cold lysis buffer and centrifuged at 3000 × g for 10 min to remove unbroken cells and wall debris. The 3000 × g supernatant was directly centrifuged at 100,000 × g for 30 min in a Beckman Optima TL ultracentrifuge. The soluble protein-containing supernatant (S100) was precipitated with 10% trichloroacetic acid and resuspended in 100 μl of lysis buffer. The membrane-containing pellet (P100) was resuspended in 100 μl of lysis buffer. Equal volumes were separated by 10% SDS-PAGE and transferred to Immobilon-PVDF membranes (Millipore). To make the cellular extracts used in Fig. 7, exponentially growing yeast cells were homogenized by vortexing with glass beads in ice-cold lysis buffer and centrifuged at 3000 × g for 10 min to remove unbroken cells and wall debris. The supernatants were collected and used directly for 8% SDS-PAGE to analyze the electrophoretic mobility of carboxypeptidase Y (CPY).FIGURE 7Co-expression of human ALG13 and ALG14 genes rescues the alg13 or alg14 glycosylation defect. Yeast strains containing GAL1p-driven ALG13 (XGY154) or ALG14 (XGY151) harboring plasmids containing the human ALG13 or ALG14 genes (pXG205 and pXG207, respectively), were grown to mid-log phase in YPA medium supplemented with galactose, shifted into YPA supplemented with galactose, or with glucose to repress expression of the ALG13 and ALG14 genes, and grown for 6∼8 h at 30°C. After three repeated passages of growth on glucose or galactose, cells were harvested and crude protein extracts were prepared as described under “Experimental Procedures.” Protein extracts (4 μg) were separated by 10% SDS-PAGE and blotted with an anti-CPY monoclonal antibody. The positions of the mature and hypoglycosylated forms of CPY lacking 1, 2, 3, or 4 N-linked glycans are indicated (37Thomas B.J. Rothstein R. Genetics. 1989; 123: 725-738Crossref PubMed Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Alg13-FLAG protein was detected by a mouse anti-FLAG monoclonal antibody conjugated to alkaline phosphatase (anti-FLAG M2-AP, Sigma) and detected directly by chemiluminescence (CDP-Star Detection Reagent, Amersham Biosciences). Mouse monoclonal anti-Dpm1 and anti-CPY (Molecular Probes) were used to detect Dpm1p and CPY. Anp1p was detected with rabbit polyclonal anti-Anp1 antibody (from Sean Munro). HA-Alg14p, Alg13-HA were detected using 12CA5 monoclonal anti-HA antibody. Primary antibody staining was followed by incubation with a secondary anti-rabbit or anti-mouse antibody conjugated to horseradish peroxidase (Amersham Biosciences) followed by chemiluminescence (ECL, Amersham Biosciences). Co-immunoprecipitation—Exponentially growing yeast cells were harvested at an A600 of 1-3 and converted to spheroplasts with lyticase. To prepare detergent extracts, spheroplasts from 5-6 Absorbance units of cells were resuspended in 500 μl of ice-cold lysis buffer (150 mm NaCl, 10 mm HEPES-KOH, pH 7.5, 5 mm MgCl2) that contained protease inhibitors and 1% digitonin as described (33Gao X.D. Dean N. J. Biol. Chem. 2000; 275: 17718-17727Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Epitope-tagged proteins in digitonin extracts were immunoprecipitated with Anti-HA affinity matrix (Roche Applied Science Co.) or Anti-FLAG M2 affinity gel (Sigma). After fractionation by SDS-PAGE, immunoprecipitates were transferred to Immobilon-PVDF membranes. To detect the FLAG-tagged proteins, the membrane was incubated with a mouse anti-FLAG monoclonal antibody conjugated to alkaline phosphatase (ANTI-FLAG M2-AP). To detect the HA-tagged protein, the membrane was incubated with a rabbit anti-HA polyclonal antibody (HA-probe Y-11, Santa Cruz Biotechnology) followed by a secondary anti-rabbit antibody conjugated to horseradish peroxidase (Cell Signaling Technologies). Analysis of GFP and mRFP Fusion Proteins—To visualize the localization of GFP and mRFP fusion proteins, cells were grown to an A600 of 1-3 in YPAD. After washing the cells with phosphate-buffered saline + 2% glucose, GFP fluorescence was imaged using the NIBA filter (Olympus), and mRFP fluorescence was imaged using the WIG filter (Olympus) on an Olympus BX50 microscope. Alg13 and Alg14 Physically Interact—Although a role for Alg13 as a glycosyltransferase involved in GlcNAc2-PP-dol synthesis requires its association with the ER membrane, it lacks any membrane-spanning domains. Interestingly, ALG14 encodes an uncharacterized membrane protein that when underproduced, leads to phenotypes similar to that of alg13 (27Chantret I. Dancourt J. Barbat A. Moore S.E. J. Biol. Chem. 2005; 280: 9236-9242Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). These data suggest a model in which these proteins pair with one another on the cytosolic face of the ER to form a functional hetero-oligomeric UDP-GlcNAc transferase that catalyzes the second step of core oligosaccharide synthesis on the ER. Thus, Alg13 and Alg14 should physically interact. To test this idea, we performed a co-immunoprecipitation assay. Yeast strains were constructed that co-express C-terminally FLAG-tagged Alg13 and N-terminally HA-tagged Alg14 (strain XGY158). This strain also contains an ALG14 allele driven by the GAL1-promoter but this allele is repressed when this strain is grown on glucose. Both of these tagged alleles are functional, expressed at physiological levels when grown on glucose, and complemented both the glycosylation defect and growth phenotype of the alg13 and alg14 mutants, respectively (data not shown). Digitonin-containing protein extracts were prepared from these strains and clarified by centrifugation at 100,000 × g to remove any nonspecific protein aggregation (see “Experimental Procedures”). Alg14 was immunoprecipitated with anti-HA antibodies, the immunoprecipitates were separated by SDS-PAGE, and blotted with anti-FLAG antibodies to determine if Alg13 precipitated with Alg14. By this assay, we found that Alg13 co-precipitated with Alg14 with high affinity and specificity (Fig. 1). Co-precipitation of Alg13 with Alg14 depended on the co-expression of Alg14-HA (Fig. 1, lane 6) and was independent of the antibodies used; immunoprecipitation of Alg13-FLAG with anti-FLAG antibodies also resulted in the co-precipitation of Alg14-HA with Alg13-FLAG (Fig. 1, lane 7). In addition, we found no evidence for the co-precipitation of other membrane proteins (e.g. Wbp1 (Fig. 1, lane 8 and data not shown), suggesting that this is a specific interaction. Taken together, these results demonstrate that these proteins physically interact. Alg13 and Alg14 Are Localized at the ER—Both Alg13 and Alg14 should be localized on ER membranes if they pair to form the UDP-GlcNAc transferase that catalyzes the formation of GlcNAc2-PP-dol on the cytosolic face of the ER. To test this idea, we constructed strains that produce GFP-tagged Alg13 and Alg14 at physiological levels to examine their localization in live cells (strains XGY167A and XGY168; see “Experimental Proced" @default.
• W2138242591 created "2016-06-24" @default.
• W2138242591 creator A5047381115 @default.
• W2138242591 creator A5050063894 @default.
• W2138242591 creator A5051362047 @default.
• W2138242591 creator A5057745536 @default.
• W2138242591 creator A5084191419 @default.
• W2138242591 date "2005-10-01" @default.
• W2138242591 modified "2023-10-01" @default.
• W2138242591 title "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" @default.
• W2138242591 cites W1484322889 @default.
• W2138242591 cites W1501577595 @default.
• W2138242591 cites W1590438213 @default.
• W2138242591 cites W1966072209 @default.
• W2138242591 cites W1983635742 @default.
• W2138242591 cites W1986616082 @default.
• W2138242591 cites W1986671043 @default.
• W2138242591 cites W2002370791 @default.
• W2138242591 cites W2006631610 @default.
• W2138242591 cites W2010168633 @default.
• W2138242591 cites W2020075737 @default.
• W2138242591 cites W2027126337 @default.
• W2138242591 cites W2028100939 @default.
• W2138242591 cites W2031894550 @default.
• W2138242591 cites W2037273428 @default.
• W2138242591 cites W2040518239 @default.
• W2138242591 cites W2047048276 @default.
• W2138242591 cites W2056199158 @default.
• W2138242591 cites W2066933680 @default.
• W2138242591 cites W2067940888 @default.
• W2138242591 cites W2071869547 @default.
• W2138242591 cites W2072500147 @default.
• W2138242591 cites W2080408951 @default.
• W2138242591 cites W2080627903 @default.
• W2138242591 cites W2118211036 @default.
• W2138242591 cites W2119097563 @default.
• W2138242591 cites W2119388326 @default.
• W2138242591 cites W2122166146 @default.
• W2138242591 cites W2129594231 @default.
• W2138242591 cites W2132933678 @default.
• W2138242591 cites W2149369491 @default.
• W2138242591 cites W2152708819 @default.
• W2138242591 cites W2159665292 @default.
• W2138242591 cites W2169333620 @default.
• W2138242591 cites W4240065971 @default.
• W2138242591 doi "https://doi.org/10.1074/jbc.m507569200" @default.
• W2138242591 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16100110" @default.
• W2138242591 hasPublicationYear "2005" @default.
• W2138242591 type Work @default.
• W2138242591 sameAs 2138242591 @default.
• W2138242591 citedByCount "103" @default.
• W2138242591 countsByYear W21382425912012 @default.
• W2138242591 countsByYear W21382425912013 @default.
• W2138242591 countsByYear W21382425912014 @default.
• W2138242591 countsByYear W21382425912015 @default.
• W2138242591 countsByYear W21382425912016 @default.
• W2138242591 countsByYear W21382425912017 @default.
• W2138242591 countsByYear W21382425912018 @default.
• W2138242591 countsByYear W21382425912019 @default.
• W2138242591 countsByYear W21382425912020 @default.
• W2138242591 countsByYear W21382425912021 @default.
• W2138242591 countsByYear W21382425912022 @default.
• W2138242591 countsByYear W21382425912023 @default.
• W2138242591 crossrefType "journal-article" @default.
• W2138242591 hasAuthorship W2138242591A5047381115 @default.
• W2138242591 hasAuthorship W2138242591A5050063894 @default.
• W2138242591 hasAuthorship W2138242591A5051362047 @default.
• W2138242591 hasAuthorship W2138242591A5057745536 @default.
• W2138242591 hasAuthorship W2138242591A5084191419 @default.
• W2138242591 hasBestOaLocation W21382425911 @default.
• W2138242591 hasConcept C138885662 @default.
• W2138242591 hasConcept C158617107 @default.
• W2138242591 hasConcept C181199279 @default.
• W2138242591 hasConcept C185592680 @default.
• W2138242591 hasConcept C190062978 @default.
• W2138242591 hasConcept C2776376580 @default.
• W2138242591 hasConcept C2777313579 @default.
• W2138242591 hasConcept C2778815778 @default.
• W2138242591 hasConcept C2779304628 @default.
• W2138242591 hasConcept C2909912800 @default.
• W2138242591 hasConcept C41895202 @default.
• W2138242591 hasConcept C55493867 @default.
• W2138242591 hasConcept C86803240 @default.
• W2138242591 hasConcept C95444343 @default.
• W2138242591 hasConceptScore W2138242591C138885662 @default.
• W2138242591 hasConceptScore W2138242591C158617107 @default.
• W2138242591 hasConceptScore W2138242591C181199279 @default.
• W2138242591 hasConceptScore W2138242591C185592680 @default.
• W2138242591 hasConceptScore W2138242591C190062978 @default.
• W2138242591 hasConceptScore W2138242591C2776376580 @default.
• W2138242591 hasConceptScore W2138242591C2777313579 @default.
• W2138242591 hasConceptScore W2138242591C2778815778 @default.
• W2138242591 hasConceptScore W2138242591C2779304628 @default.
• W2138242591 hasConceptScore W2138242591C2909912800 @default.
• W2138242591 hasConceptScore W2138242591C41895202 @default.
• W2138242591 hasConceptScore W2138242591C55493867 @default.
• W2138242591 hasConceptScore W2138242591C86803240 @default.
• W2138242591 hasConceptScore W2138242591C95444343 @default.

• next