Key points are not available for this paper at this time.
Wnt is a family of cysteine-rich secreted glycoproteins, which controls the fate and behavior of the cells in multicellular organisms. In the absence of Drosophilasegment polarity gene porcupine (porc), which encodes an endoplasmic reticulum (ER) multispanning transmembrane protein, the N-glycosylation of Wingless (Wg), one of Drosophila Wnt family, is impaired. In contrast, the ectopic expression of porc stimulates the N-glycosylation of both endogenously and exogenously expressed Wg. The N-glycosylation of Wg in the ER occurs posttranslationally, while in the presence of dithiothreitol, it efficiently occurs cotranslationally. Thus, the cotranslational disulfide bond formation of Wg competes with the N-glycosylation by an oligosaccharyl transferase complex. Porc binds the N-terminal 24-amino acid domain (residues 83–106) of Wg, which is highly conserved in the Wnt family and stimulates the N-glycosylation at surrounding sites. Porc is also necessary for the processing of Drosophila Wnt-3/5 in both embryos and cultured cells. Thus, Porc binds the N-terminal specific domain of the Wnt family and stimulates its posttranslational N-glycosylation by anchoring them at the ER membrane possibly through acylation. Wnt is a family of cysteine-rich secreted glycoproteins, which controls the fate and behavior of the cells in multicellular organisms. In the absence of Drosophilasegment polarity gene porcupine (porc), which encodes an endoplasmic reticulum (ER) multispanning transmembrane protein, the N-glycosylation of Wingless (Wg), one of Drosophila Wnt family, is impaired. In contrast, the ectopic expression of porc stimulates the N-glycosylation of both endogenously and exogenously expressed Wg. The N-glycosylation of Wg in the ER occurs posttranslationally, while in the presence of dithiothreitol, it efficiently occurs cotranslationally. Thus, the cotranslational disulfide bond formation of Wg competes with the N-glycosylation by an oligosaccharyl transferase complex. Porc binds the N-terminal 24-amino acid domain (residues 83–106) of Wg, which is highly conserved in the Wnt family and stimulates the N-glycosylation at surrounding sites. Porc is also necessary for the processing of Drosophila Wnt-3/5 in both embryos and cultured cells. Thus, Porc binds the N-terminal specific domain of the Wnt family and stimulates its posttranslational N-glycosylation by anchoring them at the ER membrane possibly through acylation. endoplasmic reticulum dithiothreitol oligosaccharyl transferase hemagglutinin Wnt is a family of cysteine-rich secreted glycoproteins and has been identified in many vertebrates and invertebrates. They have been shown to have important roles for the decision of cell fate and behavior at multiple stages during development and cancer. They bind a family of specific receptors on the cell surface (Frizzled family) and activate the cell signaling pathways to elicit their effects on, for example, gene transcription (1.Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2206) Google Scholar, 2.Polakis P. Genes Dev. 2000; 14: 1837-1851PubMed Google Scholar). One of the hallmarks of the Wnt family is the existence of 23 or 24 cysteine residues at conserved positions in the protein molecules. It has been assumed that these cysteine residues may have a critical role for folding of Wnt proteins by disulfide bond formation. However, direct evidence demonstrating that biologically active Wnt proteins contain any intra- or intermolecular disulfide bonds has not been shown. The processing and secretion of Wnt proteins was studied with cultured cells engineered to express various Wnts (3.Smolich B.D. McMahon J.A. McMahon A.P. Papkoff J. Mol. Biol. Cell. 1993; 4: 1267-1273Crossref PubMed Scopus (135) Google Scholar, 4.Burrus L.W. McMahon A.P. Exp. Cell Res. 1995; 220: 363-373Crossref PubMed Scopus (83) Google Scholar). The processing of Wnt is not efficient in most cell types, because multiple processing intermediates are present. Wnt is therefore not well secreted outside of the cells. Most of Wnt protein associates with a HSP70 protein, BiP, and is retained in the ER1(5.Kitajewski J. Mason J.O. Varmus H.E. Mol. Cell. Biol. 1992; 12: 784-790Crossref PubMed Scopus (38) Google Scholar). Meanwhile, wg mutants with lesions in the secretion and transport have been identified (6.van den Heuvel M. Harryman-Samos C. Klingensmith J. Perrimon N. Nusse R. EMBO J. 1993; 12: 5293-5302Crossref PubMed Scopus (211) Google Scholar, 7.Bejsovec A. Wieschaus E. Genetics. 1995; 139: 309-320Crossref PubMed Google Scholar, 8.Hays R. Gibori G.B. Bejsovec A. Development. 1997; 124: 3727-3736PubMed Google Scholar, 9.Dierick H.A. Bejsovec A. Development. 1998; 125: 4729-4738PubMed Google Scholar), suggesting that Wg processing and secretion is also complex in Drosophila. Caenorhabditis elegans Mom-3 appears to be necessary for Mom-2 (Wnt) processing or secretion in addition to Mom-1 (see below). Screening for genes involved in Wg signaling by Drosophila deficiency kits has also identified a new gene(s), whose product(s) is required for the processing or secretion of Wg (10.Muller H. Samanta R. Wieschaus E. Development. 1999; 126: 577-586Crossref PubMed Google Scholar). These results indicate that the processing and secretion of Wnt are complex and that a number of specific factors are involved in these events. One of the Drosophila segment polarity genes,porcupine (porc) encodes a multipass transmembrane ER protein, which is required for the normal distribution of Wg in embryos (11.Kadowaki T. Wilder E. Klingensmith J. Zachary K. Perrimon N. Genes Dev. 1996; 10: 3116-3128Crossref PubMed Scopus (252) Google Scholar). In porc mutant embryos, Wg is sequestered in its synthesizing cells and not distributed among the surrounding cells. Wg signaling components are well conserved in multicellular organisms, and porc homologs are also present in other species. C. elegans porc homolog mom-1was identified in a search for maternal genes necessary for endoderm formation (12.Rocheleau C.E. Downs W.D. Lin R. Wittmann C. Bei Y. Cha Y-H. Ali M. Priess J.R. Mello C.C. Cell. 1997; 90: 707-716Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 13.Thorpe C.J. Schlesinger A. Carter J.C. Bowerman B. Cell. 1997; 90: 695-705Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Mom-1 is necessary in Mom-2 producing cells as Porc is required in Wg-synthesizing cells. Vertebrate (mouse and Xenopus) homologs of porc have been recently identified and shown to modify the N-glycosylation of Wg and mouse Wnt proteins in cultured cells (14.Tanaka K. Okabayashi K. Asashima M. Perrimon N. Kadowaki T. Eur. J. Biochem. 2000; 267: 4300-4311Crossref PubMed Scopus (93) Google Scholar). These results demonstrate that the porc gene family encodes the evolutionary conserved ER membrane proteins involved in the processing of the Wnt family. In this study, the N-glycosylation of Wg in Drosophila S2 cells and imaginal discs was characterized in detail. We conclude that the cotranslational disulfide bond formation of Wg competes with the N-glycosylation and that Porc stimulates the posttranslational N-glycosylation by anchoring Wg at the ER membrane. Since Porc is suggested to be a member of the membrane-associated acyltransferase family, it may tether Wg to the ER membrane through acylation. Three C-terminal deleted (34, 66, and 110 amino acids) Porc mutants were generated by PCR with PorcHA28 cDNA (encoding Porc tagged with three HA epitopes at amino acid 28) as a template and the following primers: 5′-CGAGGCCTCTATTCCAGGTCATCGCCCAGCAGCAC-3′ (Δ34), 5′-CGAGGCCTCTAGGACAGGGAGTTCAATCGCCGTTT-3′ (Δ66), 5′-CGAGGCCTCTAGGCTAGGAACGCCAGCGAGATGAG-3′ (Δ110), and M13 reverse primer. The PCR products were then cloned in pCaspeR-hs. To construct a Myc epitope-tagged Wg, the DNA fragment encoding triple Myc epitopes was first PCR-amplified with pKK-1 (carrying an insert DNA encoding triple Myc epitopes) as a template and the following two primers: 5′-TACAGCTCGAGGGGTGAACAAAAGTTGATTTCTGAA-3′ and 5′-AATAGCTCGAGAAGGATCCGTTCAAGTCTTCTTCTG-3′. The PCR product was digested with XhoI and cloned in the same restriction enzyme site (at amino acid 111) of wg cDNA. This was then used as a template for PCR as described below. Deletion mutants of Myc epitope-tagged Wg were constructed by PCR with the following sets of primers: and or The PCR products were digested with and and cloned in the same restriction enzyme of pCaspeR-hs. The Wg mutants and were constructed with the PCR in and the following primers: and These wg were then cloned in pCaspeR-hs. The mutant was constructed by the fragment of mutant with that of To construct with Wg the DNA fragment encoding Wg of was PCR-amplified with the wg cDNA as the template and the following primers: and The PCR product was digested with and The DNA fragment encoding was PCR-amplified with cDNA as a template and the following primers: and The PCR product was digested with XhoI and The two PCR products were then with and DNA encoding the Myc To construct and the DNA encoding amino and of Wg were with the wg cDNA as the template and the following primers: and or PCR product was digested with XhoI and cloned at the same restriction enzyme site of the in a To construct the DNA fragment encoding amino of Wg was PCR-amplified with the wg cDNA as the template and the following primers: and The PCR product was digested with and XhoI and cloned at the same restriction enzyme of the to the DNA fragment encoding the Wg DNA encoding the were cloned in the pCaspeR-hs. Myc epitope-tagged was generated by PCR with as a template and the following primers: and The PCR product was then cloned in a DNA fragment encoding the Myc The Myc epitope-tagged cDNA was cloned in the pCaspeR-hs. PCR products were by DNA Drosophila S2 cells in were with of the DNA by to the The expression of genes was by the cells at for was to the cells at to and present the at the cells were with and then at for in the same was by of in cell and for at the of dithiothreitol was it was to the at or the addition of (at the of cells were with with of and then in the same at the cells were with and then with The cell were for and then the were The was to the and for at was then and for at The were with and then were with or The of S2 cells wg was by the cells in of and with a on The cell was then or with in the presence or absence of for on was at a of and then the were of in a proteins were with acid by in the on was and by the proteins in the were to The were with for at They were then with at the at and then with for The were at and for at The were as and the was by an The imaginal discs wg and were They were in the and then two was to the one at the of The were by by with mouse as was used as a and (porc) were a of and in S2 a of and were also The discs were in S2 and with and then in of the same was by of and for at The was then and the discs were with The cell were by with in The was as described The of and in were by a and their were This was and was by in The and of was by The same of was The embryos were with with mutant embryos as a of the P. Wieschaus Dev. Biol. Scopus Google Scholar). The embryos were to at and their were The embryos with with were with Nusse R. Dev. Biol. 1995; PubMed Scopus Google or by C. and the with The was by and In these were generated with the Perrimon N. Genetics. 1992; PubMed Google Scholar). Since Drosophila S2 cells were used to biologically active Wg Nusse R. PubMed Scopus Google and J.C. A. J. A. 1999; PubMed Scopus Google in the the processing of Wg in this cell wg is expressed in S2 three of Wg with are and Since is in the presence of of and contain In the presence of ectopic is and the is also present The and contain and three (see The is the protein in Drosophila embryos and imaginal discs Thus, Wg is at two and Porc stimulates this processing in the the of both wg and porc also the ectopic N-glycosylation of Wg at three binds the N-terminal 24-amino acid domain (residues 83–106) of Wg and stimulates the N-glycosylation at the N-glycosylation of the and mutants N-terminal and amino acids) of Myc epitope-tagged Wg was not which are in the presence of are present in Wg is at two mutants have a and proteins were by and proteins were by the processing of Wg in which the N-glycosylation were and Wg was in the absence and presence of ectopic The or residues at the number in the Wg were to the amino acid by an of Wg is with of Drosophila and mouse The conserved amino are by Myc epitope-tagged proteins of N-glycosylation and Wg of or the N-terminal amino of Wg or the Wg amino of Wg or the Wg amino of Wg were Three proteins and not are and Porc this processing the Porc and proteins and was by The cell PorcHA28 and or were with and then the were by with to the Porc is with and a not The of cell was with to that PorcHA28 is expressed at the same in cell and be the ER or the intermediates through the ER membrane. To these the cells wg were in an by to the membrane. was then with or the Wg was by and are to demonstrating that are the ER membrane These results that and are in the ER (see also shown by the is the and suggesting that of is the or the protein to We then the N-glycosylation of endogenously expressed Wg in porc and of the porc mutants with maternal to the in which gene expression is (11.Kadowaki T. Wilder E. Klingensmith J. Zachary K. Perrimon N. Genes Dev. 1996; 10: 3116-3128Crossref PubMed Scopus (252) Google Scholar). The of porc in the was by and and expression in the discs with wg expression was with Perrimon N. Development. 1993; PubMed Google Scholar). and discs were the and then the N-glycosylation of Wg was by by and are by this Thus, the N-glycosylation of in is that of in The of the to are and in and The is in porc mutant that the of the of porc results in the N-glycosylation of endogenously expressed Wg. Meanwhile, the is in Thus, the of porc stimulates the N-glycosylation of both endogenously and exogenously S2 expressed Wg. To the of Wg in Wg expressed in the imaginal discs of the was by and by that the endogenously expressed Wg is present as the in Wg with that Wg by disulfide Wg 23 cysteine residues and appears to in the imaginal discs disulfide bond formation has roles on the N-glycosylation of Wg. The N-glycosylation of Wg was in S2 cells with or has been used as a of disulfide bond formation of proteins in the ER J. A. 1992; PubMed Scopus Google Scholar, J. A. EMBO J. 1992; 11: PubMed Scopus Google Scholar, A. A. J. Cell Biol. 1993; PubMed Scopus Google Scholar, A. EMBO J. 1993; 12: PubMed Scopus Google Scholar). The cells wg and both wg and porc were with for of with in the presence of the cells in one well were with and the cell were with as The cells in two were and for in the presence or absence of The same sets of cells were also by by a in the absence of The were by and shown in in the absence of while and during the the same with ectopic the is during the and and while and during the In the cells with for is and the protein during the The of in the presence of are in the absence of This is to the of cysteine residues by a and and In the of and that the cysteine residues of Wg by disulfide bonds and are not by (see The of N-glycosylation not during the with the of Wg is the same as with ectopic the of and during the Thus, the effects of and Porc on the N-glycosylation of Wg are in the ER with of cells with for The appears during the not with demonstrating that the presence of for a the The of the same by that Wg both intra- and intermolecular disulfide bonds in S2 cells The of with and ectopic porc are that Porc is not involved in disulfide bond formation. with the Wg in the presence of both intra- and intermolecular disulfide bonds during the Porc not this Meanwhile, S2 cells with for in the ER with the and the of and this Porc has effects on the N-glycosylation of the results the The N-glycosylation of Wg occurs Wg is efficiently by a cotranslational disulfide bond formation is Porc stimulates the posttranslational N-glycosylation of Wg disulfide bond formation. The of Porc on N-glycosylation is specific to Wg, it not modify the processing of Drosophila which also both intra- and intermolecular disulfide bonds in and by the Porc is to bind Wg in the cells. To the of Porc with Wg is for its the C-terminal mutants of Porc tagged with HA epitopes were constructed and the C-terminal 66, and 110 amino of and their effects on the N-glycosylation of Wg were shown in the of and with and the of three to in the absence of ectopic This that mutants have on the N-glycosylation of Wg with PorcHA28 Porc tagged with HA that the C-terminal domain of Porc is for its We then the C-terminal deleted Porc mutants bind Wg. PorcHA28 is with Wg of the presence of contrast, the of C-terminal mutants to Wg is These results that Porc binds the Wg and that its C-terminal domain is for efficient The of mutants is not on the the that their to the ER membrane is impaired. The PorcHA28 is in embryos, it the of porc embryos both maternal and of porc as porc embryos by this mutants to the of porc embryos Thus, the of Porc with Wg appears to be necessary for the of the N-glycosylation of Wg. To the domain of Wg for its the of mutants the N-terminal and amino of were and their in S2 cells were The and mutants were tagged with triple Myc epitopes at the of Wg, because an an acid present at the C-terminal of Wg. The not are present in mutants Porc stimulates the N-glycosylation of mutants Thus, the N-terminal acid is to both the N-glycosylation of Wg and its by This construct 111) two cysteine residues in the protein and not by intermolecular disulfide bonds Thus, Porc the posttranslational N-glycosylation of Wg of the of or (see also mutants have three N-glycosylation and a site appears to be These results that Wg has two N-glycosylation at and or To the N-glycosylation of Wg, constructed Wg mutants in which the N-glycosylation were by the or residues to The N-glycosylation of these mutants is shown in The N-glycosylation of and mutants is to that of that and are not Meanwhile, and mutants in the and in the mutant the These results demonstrate that Wg is at shown in and Wg be at three to the both wg and porc are The mutant two in the presence of ectopic Thus, Wg be at and the are and both wg and porc are The of the N-terminal amino acid of and mouse Wnts that the amino acid surrounding (residues of Wg is conserved among them To this domain is a for constructed the following protein of a N-glycosylation site and Wg of the N-terminal amino of Wg Wg and amino of Wg and Wg and amino of Wg Three proteins and are and the are in the presence of ectopic Porc not the The of ectopic porc on the N-glycosylation of appears to be that on the and demonstrate that Porc binds the and not These results demonstrate that Porc binds the N-terminal 24-amino acid domain (residues 83–106) of Wg and stimulates the N-glycosylation at Porc on the N-terminal domain of Wg, which is conserved among Wnt family as described It is therefore that Porc on the processing of other Drosophila Wnt proteins in addition to Wg. To this on because its specific was Wg. In embryos at is on the of the as Nusse R. Dev. Biol. 1995; PubMed Scopus Google Scholar). In contrast, appears to be in the cell of at the of porc embryos which to expression domain Nusse R. Dev. Biol. 1995; PubMed Scopus Google Scholar). The and number of is in porc embryos, are present on the by and Thus, in porc embryos, both Wg and are not secreted the synthesizing cells. In Porc binds and stimulates its N-glycosylation in S2 cells These results therefore demonstrate that Porc on the N-glycosylation of multiple Drosophila Wnt The processing and secretion of Wnt family was shown to be in many cell various However, the of the processing and secretion of Wnts has been studied in detail. We therefore the the processing of Wnt family is and it is by Porc with Drosophila S2 cells The N-glycosylation of Wg is in S2 multiple of Wg with and two are This is to that by mouse Wnt expressed in cells J.O. J. Varmus H.E. Mol. Biol. Cell. 1992; PubMed Scopus Google Scholar). Wnt has N-glycosylation and and expressed in the three of N-glycosylation and were in Wnt with of N-glycosylation was also with other Wnts (3.Smolich B.D. McMahon J.A. McMahon A.P. Papkoff J. Mol. Biol. Cell. 1993; 4: 1267-1273Crossref PubMed Scopus (135) Google Scholar, 4.Burrus L.W. McMahon A.P. Exp. Cell Res. 1995; 220: 363-373Crossref PubMed Scopus (83) Google K. Okabayashi K. Asashima M. Perrimon N. Kadowaki T. Eur. J. Biochem. 2000; 267: 4300-4311Crossref PubMed Scopus (93) Google Scholar), this appears to be a of the Wnt family. The N-glycosylation of Wg expressed in imaginal discs is in porc mutant However, the of be in the absence of This be to the the of maternal to the N-glycosylation of Wg to Wg be by a or is not We these at this The of porc the N-glycosylation of endogenously expressed Wg which as a and by disulfide bonds These results that Porc is necessary for the efficient N-glycosylation of both endogenously and exogenously expressed Wg of the of transferase complex is at the ER membrane and with a Biochem. Res. 1999; PubMed Scopus Google Scholar). the complex is to an that is to the ER membrane to the N-glycosylation site of it is amino the J. Biol. 1993; Full Text PDF PubMed Google Scholar). the complex and one of its two the are at the ER are to in two In contrast, the other the N-glycosylation in the ER Thus, it has been that the N-glycosylation of proteins not their are However, Wg appears to be an The of complex and that Wg is necessary to be in direct with the ER membrane for the posttranslational with this the Wg at its is efficiently the secreted The N-glycosylation of Wg is by Porc which is with Porc on the of Wg, as a the N-glycosylation to the complex with The other of the posttranslational N-glycosylation a of a J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), M. J. Biol. Full Text PDF PubMed Google Scholar), an generated by J. Biol. Full Text PDF PubMed Google Scholar), and a J. Biol. Full Text PDF PubMed Google Scholar). However, the of their N-glycosylation have not been is Wg This is because Wg has 23 cysteine residues and disulfide It competes with the The N-glycosylation and disulfide bond formation was also with proteins a J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), with N-glycosylation B. J.R. EMBO J. 1996; PubMed Scopus Google Scholar), and of L.W. J. 1997; PubMed Google These proteins were at in the presence of the of ER with disulfide bond formation is and the N-glycosylation of Wg efficiently occurs cotranslationally. Porc has effects in this Since the of the disulfide bond formation of Wg not in the presence of ectopic porc Porc is not involved in this processing However, the be that Porc the N-glycosylation of Wg by the disulfide bonds formation (see below). Porc binds the N-terminal 24-amino acid (residues 83–106) domain of Wg and stimulates the posttranslational N-glycosylation at In this the is conserved in many Wnt family this has important roles for Wg processing or the of the cysteine to the secretion of Wg at the (6.van den Heuvel M. Harryman-Samos C. Klingensmith J. Perrimon N. Nusse R. EMBO J. 1993; 12: 5293-5302Crossref PubMed Scopus (211) Google Scholar). Most of the Wnt proteins Wg not have a N-glycosylation site in this the cysteine at a disulfide it is that the at be this is the Porc may disulfide bond formation at the N-glycosylation at However, the of Porc not to be to the N-glycosylation site in its shown in both and be in the presence of ectopic porc the and are These results therefore that Porc binds the N-terminal conserved domain of Wnt family and Wnt proteins at the ER membrane. The posttranslational N-glycosylation of Wnt proteins is then at the surrounding of the ER membrane by the of to the complex. with the demonstrating that Porc on the N-terminal conserved domain of the Wnt family, Porc is also to be necessary for the processing of in both embryos and cultured cells protein is in its synthesizing cells and not distributed at the in the of porc by porc embryos have the Thus, the of protein not the of This is to that with Wg (6.van den Heuvel M. Harryman-Samos C. Klingensmith J. Perrimon N. Nusse R. EMBO J. 1993; 12: 5293-5302Crossref PubMed Scopus (211) Google Scholar). However, it to be Porc is necessary for the processing of other Drosophila Wnt the following for the N-glycosylation of Wnt family. Wnt proteins disulfide bonds in and as a the N-glycosylation is Porc then binds the N-terminal specific domain of Wnt and stimulates the N-glycosylation at the surrounding sites. Porc has been recently suggested to be a member of the membrane-associated acyltransferase family K. Biochem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. PubMed Scopus Google and therefore may acid one of the amino of Wg. The of acid Wg to be with the ER and as a the of to the complex be Wg and are not secreted the synthesizing cells in the role of Porc is that of which is an acyltransferase required for the and signaling not secretion of We that the of Wg by Porc is necessary for the posttranslational N-glycosylation of Wg. It to be Porc as a acyltransferase for Wg. the folding of proteins by them in the of ER are involved in the and and for their of glycoproteins M. A. 1999; PubMed Scopus Google Scholar). Since the of Porc results in the of Wg, and may to bind Wg efficiently in the a Wg and for the The processing of the Wnt family in the is complex. It not Porc also other proteins C. elegans C.J. Schlesinger A. Carter J.C. Bowerman B. Cell. 1997; 90: 695-705Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). The of their be to Wnt signaling is at the of We for T. for Drosophila R. Nusse for and R. for and for The and were the the of the of and by the of of
Tanaka et al. (Mon,) studied this question.