Conformational Changes Induced in the Endoplasmic Reticulum Luminal Domain of Calnexin by Mg-ATP and Ca2+

Abstract

The type I membrane protein calnexin functions as a molecular chaperone for secretory glycoproteins in the endoplasmic reticulum with ATP and Ca2+ as two of the cofactors involved in substrate binding. Protease protection experiments with intact canine rough microsomes showed that amino acid residues 1-462 of calnexin are located within the lumen of the endoplasmic reticulum. Expression using the baculovirus Sf9 insect cell system of a recombinant truncated calnexin corresponding to residues 1-462 (calnexinΔTMC) revealed an association in vivo with a coexpressed secretory glycoprotein substrate, human immunodeficiency virus type I gp120. For the in vitro characterization of calnexinΔTMC, we purified this secreted form to homogeneity from the medium of Sf9 cells. We demonstrate that the properties of the purified calnexinΔTMC correspond to those of full-length calnexin in canine microsomes with at least one intramolecular disulfide bond and binding to 45Ca2+. CalnexinΔTMC underwent a marked and reversible conformational change following Ca2+ binding as measured by its resistance to proteinase K digestion of a 60-kDa fragment and also by the change from an oligomeric form of calnexinΔTMC to a monomeric form. We also found that calnexin bound Mg-ATP leading to a conformational change from a monomeric to an oligomeric form that coincided as with markedly increased proteinase sensitivity. Our results identify the luminal domain of calnexin as responsible for binding substrates, Ca2+, and Mg-ATP. Because Ca2+ and ATP are required in vivo for the maintenance of calnexin-substrate interactions, conformational changes in the luminal domain of calnexin induced by Ca2+ and Mg-ATP are relevant to the in vivo function of calnexin as a molecular chaperone. The type I membrane protein calnexin functions as a molecular chaperone for secretory glycoproteins in the endoplasmic reticulum with ATP and Ca2+ as two of the cofactors involved in substrate binding. Protease protection experiments with intact canine rough microsomes showed that amino acid residues 1-462 of calnexin are located within the lumen of the endoplasmic reticulum. Expression using the baculovirus Sf9 insect cell system of a recombinant truncated calnexin corresponding to residues 1-462 (calnexinΔTMC) revealed an association in vivo with a coexpressed secretory glycoprotein substrate, human immunodeficiency virus type I gp120. For the in vitro characterization of calnexinΔTMC, we purified this secreted form to homogeneity from the medium of Sf9 cells. We demonstrate that the properties of the purified calnexinΔTMC correspond to those of full-length calnexin in canine microsomes with at least one intramolecular disulfide bond and binding to 45Ca2+. CalnexinΔTMC underwent a marked and reversible conformational change following Ca2+ binding as measured by its resistance to proteinase K digestion of a 60-kDa fragment and also by the change from an oligomeric form of calnexinΔTMC to a monomeric form. We also found that calnexin bound Mg-ATP leading to a conformational change from a monomeric to an oligomeric form that coincided as with markedly increased proteinase sensitivity. Our results identify the luminal domain of calnexin as responsible for binding substrates, Ca2+, and Mg-ATP. Because Ca2+ and ATP are required in vivo for the maintenance of calnexin-substrate interactions, conformational changes in the luminal domain of calnexin induced by Ca2+ and Mg-ATP are relevant to the in vivo function of calnexin as a molecular chaperone. Calnexin is an integral membrane protein of the endoplasmic reticulum (ER)1 1The abbreviations used are: ERendoplasmic reticulumPMSFphenylmethylsulfonyl fluorideCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPAGEpolyacrylamide gel electrophoresisHIV-1human immunodeficiency virus type IATPγSadenosine 5’-O-(3-thiotriphosphate)BiPbinding protein. that functions as a molecular chaperone and as a constituent of the quality control apparatus of the ER (reviewed in 2Bergeron J.J.M. Brenner M. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Google Scholar). Calnexin was originally identified as a major integral membrane phosphoprotein of the ER (32Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar). Cloning of the cDNA and sequencing predicted that calnexin is a type I membrane protein with the predicted luminal domain revealing regions of sequence similarity with the major Ca2+-binding protein of the ER lumen, calreticulin. Based on the binding of 45Ca2+ to ER membrane proteins as well as the isolated protein, it was shown that calnexin is one of the two major Ca2+-binding resident proteins of the membrane of canine rough ER (32Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar; 6Cala S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Google Scholar). endoplasmic reticulum phenylmethylsulfonyl fluoride 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid polyacrylamide gel electrophoresis human immunodeficiency virus type I adenosine 5’-O-(3-thiotriphosphate) binding protein. The function of calnexin as a molecular chaperone initially arose from the demonstration that it is associated in the ER with newly synthesized major histocompatibility complex class I heavy chain and other cell surface receptors (see 2Bergeron J.J.M. Brenner M. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Google Scholar for review). Subsequently the repertoire of membrane protein substrates for calnexin was expanded to soluble monomeric secretory glycoproteins (24Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 671-676Google Scholar; 19Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Google Scholar; 33Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Google Scholar). It was observed that there is a requirement for glycosylation for proteins to bind to calnexin in vivo, because the N-linked glycosylation inhibitor tunicamycin prevented the association of newly synthesized, incompletely folded substrates with calnexin (24Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 671-676Google Scholar). In addition, deoxynojirimycin and castanospermine, inhibitors of glucosidase I and II, also abrogated the association of newly synthesized, incompletely folded viral membrane glycoproteins with calnexin. These observations led to the proposal that calnexin was a lectin with specificity for monoglucosylated N-linked oligosaccharides (15Hammond C. Helenius A. Science. 1994; 266: 456-458Google Scholar). Support for this hypothesis has come from the recent demonstrations of direct binding between a recombinant soluble calnexin and the free oligosaccharide Glc1Man9GlcNAc2 (34Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Google Scholar) and of binding of glucosylated thyroglobulin and soybean agglutinin in a cell free system.2 2W.-J. Ou, J. J. M. Bergeron, A. Parodi, and D. Y. Thomas, submitted for publication. However, in vivo calnexin can also bind non-glycosylated proteins (26Rajagopalan S. Xu Y. Brenner M.B. Science. 1994; 263: 387-390Google Scholar; 21Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 28683-28689Google Scholar; 17Kim P.S. Arvan P. J. Cell Biol. 1995; 128: 29-38Google Scholar). The N-linked oligosaccharide can also be removed by endoglycosidase H from coimmunoprecipitates of substrates bound to calnexin without disrupting their association (34Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Google Scholar). These studies indicate that the N-linked oligosaccharide may be preferred for the initial interaction of newly synthesized glycoproteins with calnexin but is not necessary to maintain the interaction. There are several different regions of calnexin that have been argued to be responsible for binding substrates. The transmembrane and immediate juxtamembrane regions of newly synthesized substrate membrane proteins have been indicated to be required for binding to calnexin (22Margolese L. Waneck G.L. Suzuki C.K. Degen E. Flavell R.A. Williams D.B. J. Biol. Chem. 1993; 268: 17959-17966Google Scholar). A role for cytosolically oriented phosphorylation of calnexin has also been proposed in the association of different allotypes of major histocompatibility complex class I heavy chains with calnexin (7Capps G.G. Ziga M.C. J. Biol. Chem. 1994; 269: 11634-11639Google Scholar). Furthermore, an ATP requirement has been demonstrated for the association of the newly synthesized soluble glycoprotein gp80 with calnexin in Madin-Darby canine kidney cells (33Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Google Scholar). However, it is clear that secretory glycoproteins can only interact directly with the luminal domain of calnexin and that membrane substrates also have the opportunity for this interaction. Taken together, these studies indicate multiple mechanisms of presentation and binding of substrates to calnexin (2Bergeron J.J.M. Brenner M. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Google Scholar). In order to establish the mechanism of association of calnexin with its substrates and to determine the cofactors involved, we have expressed a soluble recombinant derivative of calnexin in the baculovirus-infected Sf9 expression system. We demonstrate here that soluble calnexin can associate with the newly synthesized soluble glycoprotein substrate HIV-1 gp120 when they are coexpressed in Sf9 cells. We further show that soluble calnexin purified from the extracellular medium of recombinant baculovirus-infected Sf9 cells undergoes conformational changes as revealed by the formation of a Ca2+-dependent proteinase-resistant core and a Ca2+-dependent change from an oligomeric to a monomeric state, whereas Mg-ATP promoted a change from a monomeric to an oligomeric state that coincided with increased proteinase sensitivity. Finally, we show that secreted soluble calnexin is at least partially disulfide-bonded intramolecularly and can bind ATP directly but does not have detectable ATPase activity. These findings extend the functional significance of calnexin as an ATP-binding molecular chaperone with properties distinguishable from those of the ER luminal Ca2+-binding and ATP-binding chaperone, BiP. cDNA encoding a truncated canine calnexin (amino acid residues 1-462) followed immediately by an in-frame stop codon was generated by polymerase chain reaction and subsequently cloned into the transfer vector pETL1 at the BamHI site filled in with Klenow polymerase. The recombinant baculovirus was isolated by the β-galactosidase screening and used to infect Sf9 cells that were grown in Sf9000 II serum-free medium as described by 31Vialard J. Lalumire M. Vernet T. Briedis D. Alkhatib G. Henning D. Levin D. Richardson C. J. Virol. 1990; 64: 37-50Google Scholar. The sequence of the calnexin construct in the baculovirus vector was confirmed, and the following differences were observed with the published sequence (32Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar). Nucleotides 199 and 201 are to be A and A and not and as changes the codon for from to and the corresponding amino acid change is to There is also a at this in human calnexin S. Brenner M.B. J. Biol. Chem. 1993; 268: Scholar). The sequence of a of canine calnexin from this also predicted a at this we that the canine calnexin sequence is in this We also found one other with the published canine calnexin sequence at is a change of the originally predicted to an this does not change the amino acid Expression of HIV-1 gp120 in Sf9 cells by recombinant baculovirus has been described Y. L. Thomas D.Y. 1994; Scholar). Sf9 cells were with recombinant baculovirus calnexinΔTMC and grown for and acid and were to the of the used for of calnexin was to the and the was a The was using a and a with The was with the and the proteins were with a of The were with a and further purified on a protein with The proteins were with a of CalnexinΔTMC was by with a a residues of the of calnexin. calnexin but not insect calnexin. calnexinΔTMC was with of proteinase K in at for Protease digestion of calnexin was using canine ER in the of the of to the proteinase K proteins were on followed by for the purified calnexinΔTMC (see by with for the ER (see calnexinΔTMC was with in at for and to for For were with calnexin to the of The protein was on and with followed by with recombinant Sf9 cells were with for at The cells were with and in and and The cell was with protein and with as described (24Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 671-676Google Scholar). was as described by Biochem. Scholar. The 45Ca2+ was as described in I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar. was as described by Nature. Scholar. gel electrophoresis was in the of using the system. sequencing of calnexinΔTMC its was using an protein with an amino acid its sequence calnexin is predicted to be a type I integral membrane protein of the ER (32Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar). In this we the of calnexin in the ER membrane by using proteinase K digestion of canine microsomes and to in different regions of calnexin of intact microsomes revealed a proteinase-resistant that the of the protein was The is with the demonstration that in reticulum a fragment of calnexin is from digestion by S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Google Scholar). the and fragment of calnexin were by to residues of calnexin to residues only full-length calnexin in intact and proteinase this These results that in vivo calnexin is a type I membrane protein and that residues 1-462 of calnexin are oriented followed by a domain and a cytosolically oriented Because calnexin can bind membrane and soluble secretory the luminal domain is predicted to be responsible for this interaction. type I membrane proteins of the membrane domain can to their from cells. of a stop codon at immediately the predicted transmembrane domain of led to the of recombinant baculovirus and calnexinΔTMC with a of was by the with of the protein secreted at and two of and were observed for calnexinΔTMC and only the form was observed and We an that that calnexinΔTMC can interact in vivo with a viral We the association of calnexinΔTMC and that of full-length calnexin with a calnexin substrate, HIV-1 J. J. M. Bergeron, W.-J. Ou, C. Y. and D. Y. Thomas, submitted for publication. HIV-1 gp120 is from the by soluble gp120 and the membrane but in this gp120 was expressed by a codon at the site of shown in of Sf9 cells with recombinant baculovirus calnexinΔTMC HIV-1 gp120 full-length calnexin they be by of cells with and with calnexinΔTMC full-length calnexin with HIV-1 gp120 revealed their association because they be with The of calnexin in the with HIV-1 and is to the of calnexin (24Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 671-676Google Scholar). The gp120 that were associated with calnexin and were incompletely because they were not by the as has been shown for full-length and was also shown for gp120 in association with calnexinΔTMC not full-length calnexin and calnexinΔTMC to be of with a soluble glycoprotein The association of soluble calnexinΔTMC with gp120 that the luminal domain with substrates. we used this expression system to calnexinΔTMC for in vitro calnexinΔTMC was of the protein in the medium from Sf9 and it was purified to homogeneity (see The showed a for calnexinΔTMC with an molecular of on We the sequence of the residues of the purified protein as in indicate the in is to that for calnexin purified from microsomes (32Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar). The luminal domain of calnexin regions of sequence similarity with the major ER luminal protein and I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar). The results in show that the of canine ER a major protein corresponding to calnexin as well as the other major ER membrane protein We were also to show that purified soluble calnexinΔTMC can bind using the 45Ca2+ whereas in a control not full-length calnexin from ER and purified calnexinΔTMC show in their to bind to proteinase digestion was used to protein domain calnexinΔTMC was with of proteinase K in the of a fragment was generated of the proteinase a fragment of was generated and The proteinase digestion of calnexin in the ER membrane in a with and of the of and A and and of the 60-kDa fragment revealed the major sequence to be and it the sequence used to the of calnexinΔTMC with in the of also generated the 60-kDa fragment and but the sequence was one from the proteinase K Our indicate that at the and the amino of calnexin. there is a proteinase-resistant core that to maintain in the of calnexinΔTMC and calnexin to a of proteinase K A and determine the proteinase-resistant and of calnexin are calnexinΔTMC was with leading to the and was to the protein followed by the of proteinase the proteinase-resistant calnexin also the reversible conformational change of calnexinΔTMC full-length calnexin from ER and purified calnexinΔTMC can be with to the core Calnexin has residues and for canine are in from the state of secreted purified we the of the protein on and monomeric proteins with intramolecular disulfide on on the of calnexinΔTMC and calnexin in isolated ER revealed an increased and and and that calnexin at least one disulfide a we used disulfide A. Nature. and a in and Because calnexin can only at the two disulfide the found was at least one and disulfide in calnexin are of calnexinΔTMC and canine ER and and were on and and followed by with with and differences in were observed for disulfide and of calnexinΔTMC calnexin calnexinΔTMC was on to its state of A and In the of calnexinΔTMC was a A and However, with and oligomeric calnexinΔTMC were observed A and These were also by with with oligomeric calnexin with monomeric calnexin. ATP also induced the formation of calnexin the direct interaction of the with calnexinΔTMC A and The binding of to of calnexin is also with a conformational change the calnexin was on the protein as on the of the not Because and ATP induced of calnexinΔTMC, we the of calnexinΔTMC in the of purified calnexinΔTMC was with Mg-ATP and with proteinase K and calnexinΔTMC was of to the calnexinΔTMC partially proteinase K resistance and with and calnexinΔTMC was to proteinase K Because was in the reaction the that ATP is as a can be the Mg-ATP on can in the of molecular bind ATP J. Nature. and their function is by ATP S. Scholar; Science. Scholar). In vivo in Madin-Darby canine kidney ATP is required to maintain the interaction of the newly synthesized soluble glycoprotein gp80 with calnexin (33Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Google Scholar). We that purified calnexinΔTMC can bind calnexinΔTMC was with in the of and the were by followed by of ATP was and required that ATP does not form a interaction with the protein and that promoted the of ATP and the protein. The and were to for binding was the for ATP binding. also showed of ATP has also been observed for other chaperone proteins P. J. Biol. Chem. 1991; 266: Scholar). The ATP whereas as and on the binding that the disulfide in calnexin are required for ATP binding. a of the of we were to ATPase in purified We have shown that calnexin is a type I membrane protein as predicted from its that the luminal domain can bind in vivo to a secretory and that the luminal domain and full-length calnexin several calnexin was originally it was that it was by sequence to the major Ca2+-binding protein of the ER lumen, (32Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar). The of soluble calnexinΔTMC further the similarity to we that this is because of the of sequence between calnexinΔTMC and that observations on calnexinΔTMC are also directly to calreticulin. There is from in vivo experiments that calnexin can interact with newly synthesized secretory glycoproteins and membrane proteins their in the there are that membrane proteins as major histocompatibility complex class I can interact with residues within the transmembrane domain with the cytosolically domain (22Margolese L. Waneck G.L. Suzuki C.K. Degen E. Flavell R.A. Williams D.B. J. Biol. Chem. 1993; 268: 17959-17966Google Scholar; G.G. Ziga M.C. J. Biol. Chem. 1994; 269: 11634-11639Google it is clear that secretory glycoproteins can only interact with the luminal The of the protein was by and digestion of rough that the domain is in the ER lumen and interact with substrates. it is that this is the that is responsible for the and molecular chaperone for substrate We were to demonstrate by the two in Sf9 insect cells in vivo, this does interact with the secretory glycoprotein HIV-1 gp120. was by soluble truncated calnexin for the transmembrane and calnexinΔTMC, and its interaction with the soluble glycoprotein HIV-1 a substrate for full-length the of newly synthesized gp120 was found to be associated with soluble calnexin. The of the Sf9 cell to expression of proteins was because not insect cell calnexin. the studies indicated a direct interaction in vivo between calnexinΔTMC and HIV-1 gp120. for the interaction of calnexin with its substrates in its transmembrane (22Margolese L. Waneck G.L. Suzuki C.K. Degen E. Flavell R.A. Williams D.B. J. Biol. Chem. 1993; 268: 17959-17966Google Scholar; G.G. Ziga M.C. J. Biol. Chem. 1994; 269: 11634-11639Google in vivo with calnexinΔTMC and HIV-1 gp120 indicate that substrate is luminal as predicted from the studies of W.-J. Cameron P.H. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 671-676Google 33Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Google and 19Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Google Scholar. Our studies the luminal domain as the responsible for the molecular chaperone of calnexin. The from insect cells of calnexinΔTMC and its of to that this form of functions as a molecular chaperone, other properties with the full-length calnexin. calnexinΔTMC, when purified from the extracellular was found to be monomeric but to at least one intramolecular disulfide of the calnexin from to sequence the residues Based on the different of calnexin in and we that at least one disulfide bond is In the of Ca2+, calnexin and calnexinΔTMC a core to was required to maintain the core because it was by substrates by 33Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Google F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Google and 19Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Google Scholar required Ca2+ for their association with calnexin. calnexinΔTMC bound Ca2+, and Ca2+ led to the of oligomeric calnexinΔTMC to monomeric ATP led to the of monomeric calnexinΔTMC to oligomeric was by a change in the of calnexin as observed by an increased to The of Mg-ATP and Ca2+ in calnexin is of the of these in the association of incompletely folded substrate proteins with C.K. J.S. J. Cell Biol. 1991; Scholar; J. Biol. Chem. 1993; 268: Scholar). 33Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Google Scholar demonstrated that ATP is required to maintain the association of the substrate gp80 with calnexin in vivo in Madin-Darby canine kidney cells. may be by demonstration of ATP binding to calnexin and the conformational change in calnexin. The direct binding of ATP to calnexin in vitro was because adenosine and the ATP were to for binding was and binding was by However, that with free not the ATP binding activity. we that it is the disulfide that to maintain a protein in calnexin for binding and it is not free that are involved in ATP binding. The in conformational changes of calnexin as to its substrate binding to be However, it is clear that these conformational changes induced by Ca2+ and ATP are relevant to its chaperone function in The luminal of Ca2+ in the ER has been to be at of M. Nature. and the of ATP into the ER and its binding to ER proteins have also been A. J. Biol. Chem. Scholar). The requirement of and ATP for the binding of substrate proteins to calnexin that the binding of substrate proteins may conformational changes in calnexin. molecular chaperone of the also Mg-ATP. Mg-ATP and Ca2+ are also to function and conformational changes in C.K. J.S. J. Cell Biol. 1991; Scholar; S. J. Biol. Chem. 1993; 268: Scholar; A. Sci. S. A. 1994; Scholar). is a ATPase the of substrate from the chaperone C.K. J.S. J. Cell Biol. 1991; Scholar). we have been to ATPase of the purified calnexinΔTMC, this is by substrate binding to calnexin an associated protein. binding of purified calnexinΔTMC in vitro with proteins has only been demonstrated when they are glucosylated with on glycosylation J. J. Biol. Chem. However, ATPase can be in of full-length calnexin with associated secretory Ou, the that the of a interaction is required for ATPase that a protein may be required for ATPase activity. for these protein are the ER membrane proteins that we originally found associated with calnexin (32Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.-J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Google Scholar). for the of several the and a association and of with the with changes in the chaperone by ATP binding and J. Scholar; S. J. Biol. Chem. 1993; 268: Scholar; Science. 1994; Scholar; Y. 1994; Scholar). for chaperone function of calnexin (15Hammond C. Helenius A. Science. 1994; 266: 456-458Google Scholar) that associated with calnexin a as the mechanism of substrate presentation to calnexin. Calnexin and have been proposed to and in the of newly synthesized glycoproteins in vivo C. I. Helenius A. Sci. S. A. 1994; Scholar; 17Kim P.S. Arvan P. J. Cell Biol. 1995; 128: 29-38Google Scholar). ATP binding and are to the between and in protein in the ER lumen in We and Cameron for in the of We also Bell for of the and for protein We and for the insect cell at the for the of baculovirus and Dignard for the sequence of calnexinΔTMC in the recombinant was from the and