In the developing Drosophila melanogaster embryo, dorsal-ventral patterning displays an absolute requirement for the product of the essential windbeutel gene, Wind. In homozygous windbeutel mutant flies, dorsal-ventral patterning fails to initiate because of the failure of the Golgi-resident proteoglycan-modifying protein, Pipe, to exit the endoplasmic reticulum, and this leads to the death of the embryo. Here, we describe the three-dimensional structure of Wind at 1.9-Å resolution and identify a candidate surface for interaction with Pipe. This represents the first crystal structure of a eukaryotic protein-disulfide isomerase-related protein of the endoplasmic reticulum to be described. The dimeric protein is composed of an N-terminal thioredoxin domain and a C-terminal α-helical domain unique to protein-disulfide isomerase D proteins. Although Wind carries a CXXC motif that is partially surface accessible, this motif is redox inactive, and the cysteines are not required for the targeting of Pipe to the Golgi. However, both domains are required for targeting Pipe to the Golgi, and, although the mouse homologue ERp28 cannot replace the function of Wind, exchange of the Wind D-domain with that of ERp28 allows for efficient Golgi transport of Pipe. In the developing Drosophila melanogaster embryo, dorsal-ventral patterning displays an absolute requirement for the product of the essential windbeutel gene, Wind. In homozygous windbeutel mutant flies, dorsal-ventral patterning fails to initiate because of the failure of the Golgi-resident proteoglycan-modifying protein, Pipe, to exit the endoplasmic reticulum, and this leads to the death of the embryo. Here, we describe the three-dimensional structure of Wind at 1.9-Å resolution and identify a candidate surface for interaction with Pipe. This represents the first crystal structure of a eukaryotic protein-disulfide isomerase-related protein of the endoplasmic reticulum to be described. The dimeric protein is composed of an N-terminal thioredoxin domain and a C-terminal α-helical domain unique to protein-disulfide isomerase D proteins. Although Wind carries a CXXC motif that is partially surface accessible, this motif is redox inactive, and the cysteines are not required for the targeting of Pipe to the Golgi. However, both domains are required for targeting Pipe to the Golgi, and, although the mouse homologue ERp28 cannot replace the function of Wind, exchange of the Wind D-domain with that of ERp28 allows for efficient Golgi transport of Pipe. Within the developing Drosophila embryo, dorsal-ventral polarity is determined by a group of 11 genes that act to create a gradient of the NFκB homologue Dorsal within the nuclei of the embryo. Dorsal is translocated to the nucleus from the cytoplasm upon activation of the transmembrane Toll receptor by an extracellular ligand, Spätzle (1Belvin M.P. Anderson K.V. Annu. Rev. Cell Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (690) Google Scholar). Three of the eleven genes, i.e. nudel, pipe, and windbeutel, are expressed in the follicle cells. Deletion of any one of these three leads to a lethal impairment in establishing dorsal-ventral polarity (2Sen J. Goltz J.S. Konsolaki M. Schupbach T. Stein D. Development. 2000; 127: 5541-5550Crossref PubMed Google Scholar). The products of pipe and windbeutel, Pipe and Wind, are specifically required in a band of follicle cells on the ventral side of the embryo. Pipe is a Golgi-resident type II transmembrane protein with homology to mammalian heparan sulfate 2-O-sulfotransferase (3Sergeev P. Streit A. Heller A. Steinmann-Zwicky M. Dev. Dyn. 2001; 220: 122-132Crossref PubMed Scopus (20) Google Scholar, 4Kobayashi M. Habuchi H. Yoneda M. Habuchi O. Kimata K. J. Biol. Chem. 1997; 272: 13980-13985Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). It is believed that Pipe modifies an as yet unidentified proteoglycan that later participates in the proteolytic activation of Spätzle in the perivitelline space. windbeutel is expressed in all follicle cells located over the oocyte, although it seems essential only in the ventral layer (5Konsolaki M. Schupbach T. Genes Dev. 1998; 12: 120-131Crossref PubMed Scopus (59) Google Scholar). Wind is an endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; GFP, green fluorescent protein; EGFP, enhanced GFP; PDI, protein-disulfide isomerase; PDI-D, PDI-related protein containing a D-domain; MES, 4-morpholineethanesulfonic acid; PEG, polyethylene glycol.-resident, luminal protein belonging to the protein-disulfide isomerase (PDI) family, a group of proteins with varying redox and chaperone properties (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 7Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar). Although the mechanism of action remains unclear, it has recently been shown that an important role for Wind is the correct targeting of Pipe to the Golgi; windbeutel-deficient female flies show an aberrant distribution of Pipe protein, and, when expressed in COS-7 cells, Pipe is retained in the ER in a presumably inactive form. Upon simultaneous overexpression of Wind, however, a clear redistribution of Pipe to the Golgi was observed (2Sen J. Goltz J.S. Konsolaki M. Schupbach T. Stein D. Development. 2000; 127: 5541-5550Crossref PubMed Google Scholar). The mammalian homologues of Wind, ERp28 (8Ferrari D.M. Nguyen Van P. Kratzin H.D. Soling H.D. Eur. J. Biochem. 1998; 255: 570-579Crossref PubMed Scopus (75) Google Scholar) and ERp29 (9Demmer J. Zhou C. Hubbard M.J. FEBS Lett. 1997; 402: 145-150Crossref PubMed Scopus (73) Google Scholar, 10Mkrtchian S. Fang C. Hellman U. Ingelman-Sundberg M. Eur. J. Biochem. 1998; 251: 304-313Crossref PubMed Scopus (89) Google Scholar), have been found in complexes with secretory proteins (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 11Sargsyan E. Baryshev M. Szekely L. Sharipo A. Mkrtchian S. J. Biol. Chem. 2002; 277: 17009-17015Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These proteins are members of the PDI-D subfamily of PDI-related proteins, homologues of which can be found in yeast and plants as well (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). PDI-D proteins are characterized by the presence of one or two domains of roughly 100–120 residues with sequence similarity to the thioredoxin fold of PDI, followed by a novel, α-helical region at their C terminus termed the D-domain (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). The protein-disulfide isomerases are highly abundant proteins that catalyze the formation, reduction, and isomerization of disulfide bonds in substrate proteins, an activity dependent upon the presence of a redox-active CXXC tetrapeptide (7Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar). For some of these proteins it has been shown that, in addition to their redox/isomerase activities, they can also function as chaperones (12McLaughlin S.H. Bulleid N.J. Biochem. J. 1998; 331: 793-800Crossref PubMed Scopus (45) Google Scholar, 13Cai H. Wang C.C. Tsou C.L. J. Biol. Chem. 1994; 269: 24550-24552Abstract Full Text PDF PubMed Google Scholar, 14Wang L. Fast D.G. Attie A.D. J. Biol. Chem. 1997; 272: 27644-27651Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Gillece P. Luz J.M. Lennarz W.J. de La Cruz F.J. Romisch K. J. Cell Biol. 1999; 147: 1443-1456Crossref PubMed Scopus (154) Google Scholar). Recent evidence has shown that many mammalian PDI-related proteins are composed entirely of thioredoxin domains (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 16Kemmink J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. Curr. Biol. 1997; 7: 239-245Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Whereas redox function requires a CXXC motif, substrate binding requires primarily the redox-inactive b′ domain, although all domains are likely to participate. In yeasts and plants, the thioredoxin domains of PDI-D proteins are redox active (these proteins are therefore termed PDI-Dα proteins) and can rescue the otherwise lethal disruption of the pdi gene in Saccharomyces cerevisiae (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar, 17Monnat J. Neuhaus E.M. Pop M.S. Ferrari D.M. Kramer B. Soldati T. Mol. Biol. Cell. 2000; 11: 3469-3484Crossref PubMed Scopus (46) Google Scholar). Mammalian homologues, however, have a single, b-type, redox-inactive thioredoxin domain (termed PDI-Dβ proteins) (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). Here, we report on the successful crystal structure determination of a key component of the dorsal-ventral patterning pathway, which is also the first crystal structure of an ER-luminal, PDI-related protein. Through mutagenesis and functional assays, we identify a surface of the protein in the N-terminal b-domain that is likely to interact with Pipe. Replacement of a polar residue within this surface with a basic one severely impedes targeting of Pipe to the Golgi. In contrast, the nearby CXXC motif, which is redox inactive, is not required for Golgi targeting. The N-terminal domain of Wind, when expressed alone, does not promote Pipe transport to the Golgi. Furthermore, we find that although a mouse homologue of Wind, ERp28, cannot target Pipe to the Golgi, the D-domain of ERp28 can efficiently replace that of the fly protein. Thus, although the b-domain contains a site that may be involved in interaction with Pipe, the C-terminal D-domain is also required. Cell Lines, Bacterial Strains, Plasmids, and Expression Vectors— COS-7 cells were purchased from the European Collection of Animal Cell Cultures, XL1-Blue bacterial cells and the pBlueScript II plasmid were from Stratagene, pEGFP-N1 was obtained from Clontech, and the pQE-30 and pQE-60 expression vectors were from Qiagen. Antibodies and Reagents—Antibodies against Drosophila Wind were raised in rabbits against the recombinant, full-length, His6-tagged protein from Escherichia coli according to standard procedure. IgG fractions were isolated over protein A-Sepharose from Amersham Biosciences. Rabbit antibodies against the C-terminal KEEL retrieval sequence were prepared as described elsewhere (18Majoul I.V. Bastiaens P.I.H. Söling H.D. J. Cell Biol. 1996; 133: 777-789Crossref PubMed Scopus (137) Google Scholar). Goat anti-rabbit Cy3-conjugated antibody was from Jackson ImmunoResearch Laboratories. For mutagenesis, the QuikChange site-directed mutagenesis kit from Stratagene was used. Cell culture media were from Sigma, and the Pefabloc SC protease inhibitor was purchased from Biomol. cDNA Cloning and Expression Vector windbeutel cDNA the Wind was by from a cDNA of M. of For bacterial a product Wind, was an N-terminal a This is to as Wind was prepared by a of Wind the of ERp28 cDNA was by from cells. For expression in mammalian cells, windbeutel was the of pEGFP-N1 that a the enhanced green fluorescent protein of Wind was for Wind and were and the QuikChange site-directed mutagenesis of Wind the N-terminal thioredoxin domain alone, with or an retrieval sequence and were by that a was or an the KEEL retrieval was prepared by the sequence for the C-terminal domain of mouse ERp28 with with that of the b-domain of Wind the Wind D-domain for that of mouse of in an Pipe pipe was in three of the of pipe and an of the of were from a Drosophila cDNA of of pipe was obtained by of from flies from M. for were pBlueScript of and of was and used to and a and an The site was later to the sequence to pipe was by a from Pipe Expression from this a Pipe protein to the terminus of the Cell and cells were in and with and at and For cells were by in a II and on with Wind antibody was used at a and of antibody was with a of a anti-rabbit Cy3-conjugated of and was on an with with an of and a of and and of and Expression and Wind protein as well as and were expressed from and in E. coli XL1-Blue cells by of an culture for at with The proteins were by of cells in Pefabloc SC protease inhibitor followed by addition of to and over a protein was with of and and and with of and and in of and protein was against and to and at was by and was and all of the of Wind 272: Scopus Google Scholar) on a and an of protein by on a was The protein was and in and at of three of of protein were for and were by the at The were by of Wind in with a containing MES, and MES, and was used as the The was prepared by with a well of MES, and and in MES, and for Collection and were at The were by 1997; PubMed Scopus Google Scholar), 1997; PubMed Scopus Google Scholar), and D Biol. 1994; PubMed Scopus Google Scholar). The to group with two in the The were on a at the in at the The and were used for of the and structure and or are in as for and for are in as for and for in a and structure was by The of three was and by a of and by the M. E. for Scholar) with the to 2002; Scopus Google Scholar) was used for and was used to by and The was by with the of D.G. D Biol. 2001; PubMed Scopus Google Scholar). the was by A. Biol. 1999; PubMed Scopus Google Scholar). the was used for to and The structure was with D Biol. 1997; PubMed Scopus Google Scholar) an was with J. Biol. 1999; PubMed Scopus Google Scholar), and only for which was were the The structure was with J.M. J. Google Scholar). the residues have within the The were with J. Google Scholar), and D Biol. 1994; PubMed Scopus Google Scholar). and structure have been in the with the Drosophila Wind as a and is composed of two domains by a of 11 residues The N-terminal b-domain has residues and an fold with the of structure The of the with the of have a a of all by the This fold is for protein-disulfide isomerase and proteins D.M. H. J. Mol. Biol. PubMed Scopus Google Scholar, J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. 1996; PubMed Scopus Google Scholar, J. Dijkstra K. M. E. Nilges M. Darby N.J. J. Biomol. 1999; PubMed Scopus Google Scholar). The C-terminal domain is composed of residues and has a termed the D-domain (6Ferrari D.M. Söling H.D. Biochem. J. 1999; 339: 1-10Crossref PubMed Scopus (441) Google Scholar). This which has all in represents a fold unique to PDI-D proteins. The residues in were from to be for the first and for the The N-terminal sequence not be in the crystal structure of Wind a and by a of the N-terminal with by residues within the The is and primarily of residues to side of residues within and and residues within and which are involved in a of as well as to from residues within and and the of the with mammalian PDI-D proteins) and of the and a of and of and the and residues as well as the and The side of in is involved in a of with and as well as with of and the in the In bonds can be for residues within and The surface is which to of the surface of the protein. a with the 11 11 the of the in the of this The to as the which is in is by residues from the and and residues from the of and the with residues within and at the motif at the the to the and and may be for with substrate cysteines within the with the not not only for the of also for the first of the motif of one of the two that these are of the of the domains of the two of their three-dimensional with for of and for the b-domain and the The in of of the with a of the within the the two domains of both not be a of the which be to the region the and C-terminal domains and primarily to the and of this is that, in the the D-domain in a that the the of the D-domain from of of from a of to within of the from of Wind is also by 272: Scopus Google Scholar) in and in and not of the His6-tagged protein, which a of The the in with Pipe, Wind for Pipe of the of Wind the an to partially an with on the surface the and on of the The surface the which a of three and in PDI-Dβ homologues and the presence of a This site is to a PDI-related proteins of the of In Wind, a which is for residues in redox-active thioredoxin residues the may be involved in interaction with Pipe, we Pipe and Wind in COS-7 cells in which Wind was expressed as or with a within the is surface at the of the is at from the Replacement with was to the surface of the the of the protein. Pipe to exit the ER when with although the mutant is highly expressed and retained in the ER and that the surface the surface of the by the is required for Pipe from the can the that the from an of Wind to because a to which is to Wind Furthermore, the were a of the of Wind to one that the expression of a mutant that or Pipe this we a Wind which was on the of the crystal structure to fails to in although it is when with Pipe in cells, to catalyze the of Pipe to the Golgi This that a can in or that dimeric Wind is not for Pipe Thus, the of function of the mutant is likely to a of substrate binding any impairment to the and of Wind for of Pipe, the of the Wind D-domain by of Mammalian ERp28 the b-domain be to Pipe to the Golgi. this we the of Pipe in cells a Wind the D-domain residues as well as in cells an to a KEEL retrieval sequence In COS-7 cells, although the was retained in the ER to of a retrieval the was efficiently retained and However, in was the of Pipe to the Golgi and This that the D-domain also participates in the or the transport of Pipe. In we the of the mouse homologue of Wind, ERp28, to replace Wind in targeting Pipe to the Golgi. In ERp28 to promote of Pipe to the Golgi not In contrast, when a the N-terminal domain of Wind to the D-domain of ERp28 was an Golgi distribution of Pipe was observed the of by not from that by Wind. that, although ERp28 cannot replace Wind in Pipe to the Golgi, the D-domain of ERp28 can efficiently replace the domain of Wind an of only for the The in Wind for Pipe and has a tetrapeptide at terminus This which does not in PDI-Dβ homologues in is located within the from and the site of the and from the motif of the In PDI, the tetrapeptide at the terminus of which is believed to the of the active site T. Darby N.J. Creighton T.E. 1996; PubMed Scopus Google Scholar). However, also for redox-active as was for the protein M. J. EMBO J. 2002; PubMed Scopus Google Scholar) and proteins that a thioredoxin fold J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. A. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. 1998; PubMed Scopus Google Scholar). The that the D-domain of ERp28 can replace that of Wind, although ERp28 does not promote Pipe in a substrate binding site to The surface is highly is the motif in Wind located the cysteines of this motif are required for Pipe transport to the Golgi has we a mutant of Wind both cysteines of the site and to Pipe to the Golgi. in of Pipe was observed for with COS-7 cells with windbeutel, and In cells Wind, Pipe was retained in the ER as (2Sen J. Goltz J.S. Konsolaki M. Schupbach T. Stein D. Development. 2000; 127: 5541-5550Crossref PubMed Google Scholar) we found that Wind is to the disulfide of in in and to a of S. cerevisiae and in the of this that Wind a redox/isomerase that the tetrapeptide of Wind is redox inactive and not required for the correct targeting of Pipe to the Golgi. and to PDI-related has a redox inactive thioredoxin domain sequence and similarity with both the redox-active and and redox-inactive and domains of Although the have from the and the thioredoxin domain sequence retained only a of homology to these domains it is that their presence is essential not only for the redox also for the chaperone function of in the (7Freedman R.B. Klappa P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar, P. Ruddock L.W. Darby N.J. R.B. EMBO J. 1998; PubMed Scopus Google Scholar). of both the and the b-domain of have been J. Darby N.J. Dijkstra K. Nilges M. Creighton T.E. 1996; PubMed Scopus Google Scholar, J. Dijkstra K. M. E. Nilges M. Darby N.J. J. Biomol. 1999; PubMed Scopus Google Scholar). on the b-domain of Wind homology with the of and homology with the For the the homology is similarity for all three In contrast, the of homology the and of is Wind mammalian homologue a of homology to the and of these domains with The residues are primarily within the of the fold or on of and are likely to a of the of the thioredoxin domains of Wind and of and for Wind and the and of PDI, For the and alone, a of was on this the structure of the N-terminal domain of Wind may be to that of the b-domain the the thioredoxin domains of Wind and is the presence in Wind of an of function in the and This is surface the and is partially in PDI-Dβ proteins In the b-domain of Wind, the and domains of and proteins as well as in a in is and This has been to of a site to the active site in proteins K. A. J. Mol. Biol. 1999; PubMed Scopus Google Scholar), and is important for the of the thioredoxin In Wind as this surface some to a of and is partially in PDI-Dβ proteins and may be involved in substrate The however, a Thus, the structure of the b-domain of Wind seems to be of the and of the domains of ERp29 were E. Baryshev M. Sharipo A. Ingelman-Sundberg M. Mkrtchian S. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Although the thioredoxin domain with the thioredoxin domain of Wind for of the by and that by crystal structure are on the of the thioredoxin Furthermore, in the residue to was found to be we were to crystal structure of the D-domain over the one by In the and are from the of the in Wind and have a of their For Wind, a residues within the of the domain and is to have shown that the D-domain of mouse ERp28 can replace the domain of Wind when expressed as a protein, that the structure of the D-domain of ERp28 of is likely to that of the D-domain of Wind. However, it is clear that the D-domain does some which is not only by the in of the of the two also by the of the two to when the two domains are the similarity Wind and ERp29 some in and structure cannot be is a key in dorsal-ventral patterning in of function was by Stein and (2Sen J. Goltz J.S. Konsolaki M. Schupbach T. Stein D. Development. 2000; 127: 5541-5550Crossref PubMed Google Scholar), found a requirement for Wind in the transport of the Golgi-resident Pipe protein from the ER to the Golgi. The surface of Wind a by of the can show that of a polar residue at the of the with a basic one which does not in Pipe is located in a of residues and in the to the of the active site tetrapeptide in In it seems that all three as well as nearby residues and within the may be involved in the of Pipe. C. S. and D. M. in Wind is required for Pipe remains to be Wind which primarily as a in Pipe in This may be to a of or may not be required at a interaction with Pipe substrate binding may at in the surface of Wind in the of also with of the the in In Pipe a region to be within the luminal domain a of basic residues from located with this were to a binding surface for Wind, the be to that for the interaction of the reticulum protein with the protein L. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. Biol. 1998; PubMed Scopus Google Scholar). may that Pipe in residues to of Wind within or the and, in with on the surface This interaction may with the of Pipe in the ER and basic the Pipe and it transport functional the structure of the PDI-D protein Wind is the to to the of the and of that many of the PDI-related proteins have in with the b-domain of Wind is the of their thioredoxin fold the structure of Wind, in with a of may also binding properties and of and in and PDI-related proteins. are to for with and for of the with
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