Identification of an N-domain Histidine Essential for Chaperone Function in Calreticulin (2003)

Calreticulin is an endoplasmic reticulum (ER) luminal Ca2+-binding chaperone involved in folding of newly synthesized glycoproteins via the “calreticulin-calnexin cycle.” We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt-/- cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders. Calreticulin is an endoplasmic reticulum (ER) luminal Ca2+-binding chaperone involved in folding of newly synthesized glycoproteins via the “calreticulin-calnexin cycle.” We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt-/- cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders. The endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumMDHmalate dehydrogenaseANS8-anilino-1-naphthalene-sulfonic acidHAhemagglutininMOPS4-morpholinepropanesulfonic acid.1The abbreviations used are: ERendoplasmic reticulumMDHmalate dehydrogenaseANS8-anilino-1-naphthalene-sulfonic acidHAhemagglutininMOPS4-morpholinepropanesulfonic acid. plays an essential role in a variety of cellular processes, including Ca2+ homeostasis, protein and lipid synthesis, and post-translational modification and folding of membrane-associated and secreted proteins (1.Baumann O. Walz B. Int. Rev. Cytol. 2001; 205: 149-214Crossref PubMed Scopus (328) Google Scholar). The ER ensures that only correctly folded proteins proceed through the secretory pathway and directs misfolded proteins to ER-associated degradation (2.McCracken A.A. Brodsky J.L. BioEssays. 2003; 25: 868-877Crossref PubMed Scopus (194) Google Scholar, 3.Cabral C.M. Liu Y. Moremen K.W. Sifers R.N. Mol. Biol. Cell. 2002; 13: 2639-2650Crossref PubMed Scopus (91) Google Scholar). The lumen of the ER is a dynamic environment that contains numerous molecular chaperones and Ca2+-binding proteins that are designed for these tasks. Molecular chaperones are proteins that bind to misfolded/unfolded proteins in a transient manner to assist in their folding. endoplasmic reticulum malate dehydrogenase 8-anilino-1-naphthalene-sulfonic acid hemagglutinin 4-morpholinepropanesulfonic acid. endoplasmic reticulum malate dehydrogenase 8-anilino-1-naphthalene-sulfonic acid hemagglutinin 4-morpholinepropanesulfonic acid. Calreticulin is a Ca2+-binding chaperone that resides in the lumen of the ER and is involved in modulation of Ca2+ homeostasis and in the folding of newly synthesized glycoproteins via the “calreticulin-calnexin cycle” (4.Schrag J.D. Procopio D.O. Cygler M. Thomas D.Y. Bergeron J.J. Trends Biochem. Sci. 2003; 28: 49-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 5.Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (531) Google Scholar, 6.Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1645) Google Scholar, 7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). Calreticulin and calnexin are both ER lectins, which bind transiently to virtually all newly synthesized glycoproteins (5.Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (531) Google Scholar, 6.Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1645) Google Scholar, 7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). Chaperone-assisted protein folding has been studied extensively using Escherichia coli GroEL heat shock proteins, which are cytoplasmic (8.Feltham J.L. Gierasch L.M. Cell. 2000; 100: 193-196Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Numerous studies have been carried out on ER-associated chaperones (2.McCracken A.A. Brodsky J.L. BioEssays. 2003; 25: 868-877Crossref PubMed Scopus (194) Google Scholar, 3.Cabral C.M. Liu Y. Moremen K.W. Sifers R.N. Mol. Biol. Cell. 2002; 13: 2639-2650Crossref PubMed Scopus (91) Google Scholar, 4.Schrag J.D. Procopio D.O. Cygler M. Thomas D.Y. Bergeron J.J. Trends Biochem. Sci. 2003; 28: 49-57Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 5.Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (531) Google Scholar, 6.Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1645) Google Scholar, 7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar); yet, the molecular features of calreticulin that confer its chaperone function have not yet been determined (7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). Three distinct structural domains have been identified in calreticulin: the amino-terminal, globular N-domain; the central P-domain; and the carboxyl-terminal C-domain (7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar). NMR (9.Ellgaard L. Riek R. Herrmann T. Guntert P. Braun D. Helenius A. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3133-3138Crossref PubMed Scopus (155) Google Scholar), modeling (10.Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (360) Google Scholar), and biochemical studies (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar) indicate that the globular N-domain and the “extended arm” P-domain of calreticulin may form a functional protein-folding unit (10.Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (360) Google Scholar). This region of calreticulin contains a Zn2+ binding site and one disulfide bond, and it may also bind ATP (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar, 13.Andrin C. Corbett E.F. Johnson S. Dabrowska M. Campbell I.D. Eggleton P. Opas M. Michalak M. Protein Expression Purif. 2000; 20: 207-215Crossref PubMed Scopus (16) Google Scholar, 14.Corbett E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar). When calreticulin binds Zn2+, it undergoes dramatic conformational changes (15.Khanna N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar). Chemical modification of calreticulin has revealed that four histidines located in the N-domain of the protein (His25, His82, His128, and His153) are involved in the Zn2+ binding (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar). The Zn2+-dependent conformational change in calreticulin affects its ability to bind to unfolded protein/glycoprotein substrates in vitro Y. Y. J. 1999; PubMed Scopus Google Scholar), that conformational changes in calreticulin may its chaperone function. The role of the Zn2+ binding histidine in calreticulin function is not Calreticulin is and cells from calreticulin have Ca2+ homeostasis and protein folding and (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar, R. Ahsan I. Mesaeli N. Michalak M. Biochem. 2003; PubMed Scopus Google Scholar). The of calreticulin-deficient cells an for of the molecular with calreticulin function in the lumen of the In mutants of calreticulin and reconstituted the ER lumen of calreticulin-deficient In calreticulin-deficient folding of the is altered, which its ability to Ca2+ release (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). the reestablishment of bradykinin-dependent Ca2+ release from the ER was used as a marker for calreticulin function. Zn2+-dependent conformational changes in calreticulin are for its with proteins Y. Y. J. 1999; PubMed Scopus Google Scholar), on the role of histidine in calreticulin function. We that of the histidine in the N-domain of calreticulin only His153 is essential for its function. of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders. malate dehydrogenase 8-anilino-1-naphthalene-sulfonic acid and from was from and molecular form was from and from was from of the and coli of wild type calreticulin was and and of to calreticulin in the calreticulin was and and of of the a hemagglutinin was to the of calreticulin to was carried out using a PubMed Scopus Google Scholar, Google Scholar) using thermal and biochemical and calreticulin mutant proteins in coli calreticulin of was of The histidine to or histidine mutants and and to or histidine mutations wild type calreticulin and mutants are as and and Cell and Ca2+ type and calreticulin-deficient used in (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). in a environment in crt-/- cells with wild type or mutant carried out using and with The wild type calreticulin or histidine mutants and of cytoplasmic Ca2+ with the Ca2+ (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Ca2+ release from was with and in a (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). cells with and with and on and to with was used as the on in with and with and with was used as in and with a with a Expression and of in coli cells in of to the by the of the of proteins with of for for The was in a and in the by for proteins by in of coli with the with a and the and with a and proteins by a and proteins in a and of the protein was to by Protein was determined by a amino acid or by using protein using as a Biochem. PubMed Scopus Google Scholar). was with of wild type and mutant calreticulin and in and for for was from to the of the was and The protein was in a Y. Y. J. 1999; PubMed Scopus Google Scholar) by the of wild type or mutant calreticulin Protein aggregation was by to was using a with a the and to and and in a Three calreticulin wild type and mutant proteins was used for in a and (15.Khanna N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar). The was to and the of was to The of Zn2+ on the of protein was a of analysis was using a to an and by as E.F. Oikawa K. P. Kay C. Bergeron Thomas D.Y. Krause K.-H. Michalak M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The for by the analysis of and J. 20: PubMed Scopus Google Scholar). of wild type and mutant calreticulin in coli with E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar). and the was by the of the PubMed Scopus Google Scholar). The proteins in and with Expression of in N-domain of calreticulin contains histidine which are involved in Zn2+ binding to the protein (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar). Zn2+ binding to calreticulin significant conformational which the of calreticulin with substrates (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar, 14.Corbett E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar, N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar, Y. Y. J. 1999; PubMed Scopus Google Scholar, S. K. C. Michalak M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. 2001; PubMed Scopus Google Scholar). the role of the N-domain histidines in calreticulin function, His25, His82, His128, and His153 to a of these mutants and as and cells with these to wild type calreticulin or the histidine mutants and of proteins, the was the We have that of a not calreticulin function (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar, S. M. Nakamura K. C. Michalak M. Demaurex N. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, B. R. M. Nakamura K. R. Michalak M. T. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). analysis that all of the cells calreticulin of of protein was in cells the and His128 mutants and to cells with a of carried out analysis of the to that the calreticulin was to the of the and and the protein was to an a the ER in all as by with and calnexin not Ca2+ in Calreticulin have that calreticulin-deficient cells have bradykinin-dependent Ca2+ which is by of calreticulin (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). This is in the of the not it is to bind to (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). of bradykinin-dependent Ca2+ release from the ER an to the function of calreticulin and calreticulin mutants in crt-/- bradykinin-dependent Ca2+ release in cells the calreticulin mutants We these with the as a and transient in the cytoplasmic Ca2+ in wild type cells but not in crt-/- cells (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). the of calreticulin in crt-/- cells bradykinin-dependent Ca2+ release crt-/- Ca2+ release was in the and Expression of the His25, His82, and His128 mutants bradykinin-dependent Ca2+ release whereas of the His153 mutant did This that His153 play an essential role in and chaperone function. His153 of and to the role of His153 in the chaperone of an in vitro used by Y. Y. J. 1999; PubMed Scopus Google Scholar). This of a and IgY, a Y. Y. J. 1999; PubMed Scopus Google Scholar). is to aggregation as by and it has been used as a in aggregation and with molecular chaperones J. PubMed Scopus Google Scholar, L. S. J. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. A. M. B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). has that calreticulin or aggregation in vitro Y. Y. J. 1999; PubMed Scopus Google Scholar). the role of histidine in calreticulin function, the of the His25, His82, His128, and His153 mutants in of thermal aggregation of and IgY, in In to the proteins in coli and that of the proteins on a was was to it to form that be by Y. Y. J. 1999; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, L. S. J. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. A. M. B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). aggregation was in the of of wild type calreticulin Y. Y. J. 1999; PubMed Scopus Google Scholar). the of the His153 calreticulin mutant to or did not aggregation In mutant prevented aggregation of to wild type protein In the and His128 mutants also and and their was in the these mutants all prevented thermal aggregation of to wild type calreticulin not the of calreticulin mutants in aggregation of a The of the of calreticulin on aggregation are by Y. Y. J. 1999; PubMed Scopus Google Scholar). calreticulin prevented aggregation of The His25, His82, and His128 mutants also in aggregation of in with the of the the His153 mutant did not aggregation of of Calreticulin modeling of the of on the of calnexin J.D. Bergeron Y. S. M. Thomas D.Y. Cygler M. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), that His153 is located in a to a binding site This region is involved in Zn2+ binding to calreticulin (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar), mutation of His153 residue may Zn2+-dependent conformational changes in the its with its substrates Y. Y. J. 1999; PubMed Scopus Google Scholar). carried out and analysis and of wild type and mutant the of calreticulin and the calreticulin The of a protein is by the of and by changes in the from of the the on changes in the protein is is by the the of for wild type calreticulin and for calreticulin mutants Zn2+ that in the of Zn2+ both wild type calreticulin and the His153 mutant have an of the of wild type calreticulin was that for the His153 mutant This that the mutant protein has to the In the of of Zn2+, was a significant in the of from wild type calreticulin and indicating Zn2+-dependent conformational changes in the In the His153 calreticulin mutant only changes in as a function of Zn2+ and This that the of Zn2+ on calreticulin and conformation is in the of changes in also and significant wild type calreticulin and calreticulin His153 mutant and changes in His153 analysis of wild type calreticulin and His153 His153) mutant was carried out as analysis was carried out in the and of of wild type His153 mutant. of Zn2+-dependent changes in of wild type and His153 mutant of calreticulin are in D. are changes in of wild type calreticulin or His153 mutant The was to and the of was to has been used extensively to on the of When is to a its is and is by a wild type and His153 mutant calreticulin a of the from to and the of Zn2+ a from to The of was not the of wild type calreticulin a in with for the His153 mutant calreticulin The of Zn2+ to for wild type calreticulin with only for His153 mutant This that the His153 calreticulin mutant has a wild type the in and conformational the wild type and His153 mutant carried out analysis of the that the of calreticulin and the His153 mutant are in The for structural the for both proteins as In the and for the proteins for wild type calreticulin and for the His153 mutant. This in in the mutant protein the of and that mutation of His153 has a significant on the of proteins a conformational change the of Zn2+ with a in the of The of Zn2+ to wild type calreticulin in and an in and In the of Zn2+ to the His153 mutant calreticulin in and a significant in structural analysis of wild type calreticulin and calreticulin His153 mutant in the and of Zn2+ carried out in the of a and type type in a conformational calreticulin and calreticulin mutants using We have that the of calreticulin to is an of its folding E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar). the of His153 mutation on proteins with for to and by that a significant of wild type calreticulin was to for to In the His153 calreticulin mutant was and of with the This to that mutation of His153 in calreticulin to significant of its is that the ER plays a role in the folding of secretory and membrane-associated proteins and The ER contains of molecular including disulfide the and proteins, and the calreticulin and calnexin Y. Cell Biol. 1999; PubMed Scopus Google Scholar). it has been to the function of these chaperones a structural but the of calreticulin-deficient cells has with a to the molecular features of calreticulin that are for its chaperone function. In reconstituted the ER of calreticulin-deficient cells with calreticulin mutants and a functional analysis in This was by in vitro of the of calreticulin mutants to the of mutations on the protein and function. We on the N-terminal histidine of their role in Zn2+ binding to calreticulin Zn2+-dependent conformational changes are for Y. Y. J. 1999; PubMed Scopus Google Scholar). mutation of one histidine residue calreticulin function as by its ability to the crt-/- cells have bradykinin-dependent Ca2+ release (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). In these not bind to the the is folded (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). Bradykinin-dependent Ca2+ release from the ER was rescued by with wild type calreticulin (11.Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar) and calreticulin with His25, His82, or In Ca2+ release in crt-/- cells was not rescued by with the His153 mutant. on the of calreticulin and the calreticulin mutants that wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of and IgY, whereas the His153 mutant did We conclude that His153 is essential for the chaperone function of the molecular the loss of chaperone in the His153 used and studies to the His153 mutation affects the of The of the His153 mutant that conformational changes may have in the mutant Zn2+-dependent conformational a of calreticulin (15.Khanna N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar), also in the His153 mutant. This with that the N-domain histidine are involved in Zn2+ binding to calreticulin (12.Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar). The analysis that the His153 mutant has a wild type analysis revealed that the His153 mutant from wild type calreticulin in and the studies that the His153 mutant is to that the wild type the His153 mutation in changes in calreticulin and these its ability to function as a molecular We have the of calreticulin (10.Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (360) Google Scholar) on for calnexin J.D. Bergeron Y. S. M. Thomas D.Y. Cygler M. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and NMR for the P-domain of calreticulin L. Riek R. Braun D. Herrmann T. Helenius A. Wuthrich K. FEBS Lett. 2001; PubMed Scopus Google Scholar). This an for of and for the role of the region in protein folding The of calnexin that it of a globular and an P-domain an the J.D. Bergeron Y. S. M. Thomas D.Y. Cygler M. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The P-domain of calreticulin also an (9.Ellgaard L. Riek R. Herrmann T. Guntert P. Braun D. Helenius A. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3133-3138Crossref PubMed Scopus (155) Google Scholar). The is an that a binding including the site J.D. Bergeron Y. S. M. Thomas D.Y. Cygler M. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of N-terminal globular and central P-domain that the N-terminal is to form a globular (10.Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (360) Google Scholar). with the form a folding unit in calreticulin (10.Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (360) Google Scholar, K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.-H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). of has to the of the N-terminal histidine in His25, His82, and His128 are all on the of the globular N-domain of the from the of the P-domain to be located from the binding region in their mutation or has on function as a molecular In His153 is calreticulin is on the of a that is of a located the the globular N-domain and the “extended arm” P-domain the His153 is to be with the of the This may the of the binding Calreticulin and calnexin are chaperones with a for are a of protein substrates that are to calreticulin or calnexin (7.Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (661) Google Scholar), and it is not is that the histidine in the of the proteins play a are histidine in the N-terminal globular of calnexin J.D. Bergeron Y. S. M. Thomas D.Y. Cygler M. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, I. D. J.J. D. A. D. Thomas D.Y. Bergeron J. Biol. Chem. Full Text PDF PubMed Google Scholar), and only of these are in The are His128, which on calreticulin function in and His153 in the His153 is in the amino acid that residue in the of in are amino acid that form a not in calreticulin J.D. Bergeron Y. S. M. Thomas D.Y. Cygler M. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The in calnexin in calreticulin and may have is that the that contains the His153 residue may to of In that a single amino acid mutation in an ER luminal affects protein folding. that a mutation as in a of protein folding. This has in of protein folding We S. for