BiP, a Major Chaperone Protein of the Endoplasmic Reticulum Lumen, Plays a Direct and Important Role in the Storage of the Rapidly Exchanging Pool of Ca2+

The activity of BiP, the major chaperone of the endoplasmic reticulum (ER) lumen, is known to be Ca2+-regulated; however, the participation of this protein in the ER storage of the cation has not yet been investigated. Here such a role is demonstrated in human epithelial (HeLa) cells transiently transfected with the hamster BiP cDNA and incubated in Ca2+-free medium, as revealed by two different techniques. In the first, co-transfected aequorin was employed as a probe for assaying either the cytosolic of the mitochondrial free Ca2+ concentration. By this approach higher Ca2+ release responses were revealed in BiP-transfected cells by experiments in which extensive store depletion was induced either by repetitive stimulation with inositol 1,4,5-trisphosphate-generating agonists or by treatment with the Ca2+ ionophore, A23187. In the second technique the cells were loaded at the equilibrium with 45Ca, and the release of the tracer observed upon treatment with thapsigargin, a blocker of the ER Ca2+ ATPases, was larger in BiP-transfected than in control cells. The latter results were obtained also when BiP was overexpressed not via transfection but as a response to ER stress by tunicamycin. These results are sustained by increases of the ER Ca2+ storage capacity rather than by artifacts or indirect readjustments induced in the cells by the overexpression of the chaperone since (a) the exogenous and endogenous BiP were both confined to the ER, (b) the expression levels of other proteins active in the ER Ca2+ storage were not changed, and (c) effects similar to those of wild type BiP were obtained with a deletion mutant devoid of chaperone activity. The specificity of the results was confirmed by parallel 45Ca experiments carried out in HeLa cells transfected with two other Ca2+-binding proteins, calreticulin and CaBP2(ERp72), only the first of which induced increases of Ca2+ capacity. We conclude that BiP has a dual function, in addition to its chaperone role it is a bona fide ER lumenal Ca2+ storage protein contributing, under resting cell conditions, to around 25% of the store, with a stoichiometry of 1–2 moles of calcium/mole of BiP. The activity of BiP, the major chaperone of the endoplasmic reticulum (ER) lumen, is known to be Ca2+-regulated; however, the participation of this protein in the ER storage of the cation has not yet been investigated. Here such a role is demonstrated in human epithelial (HeLa) cells transiently transfected with the hamster BiP cDNA and incubated in Ca2+-free medium, as revealed by two different techniques. In the first, co-transfected aequorin was employed as a probe for assaying either the cytosolic of the mitochondrial free Ca2+ concentration. By this approach higher Ca2+ release responses were revealed in BiP-transfected cells by experiments in which extensive store depletion was induced either by repetitive stimulation with inositol 1,4,5-trisphosphate-generating agonists or by treatment with the Ca2+ ionophore, A23187. In the second technique the cells were loaded at the equilibrium with 45Ca, and the release of the tracer observed upon treatment with thapsigargin, a blocker of the ER Ca2+ ATPases, was larger in BiP-transfected than in control cells. The latter results were obtained also when BiP was overexpressed not via transfection but as a response to ER stress by tunicamycin. These results are sustained by increases of the ER Ca2+ storage capacity rather than by artifacts or indirect readjustments induced in the cells by the overexpression of the chaperone since (a) the exogenous and endogenous BiP were both confined to the ER, (b) the expression levels of other proteins active in the ER Ca2+ storage were not changed, and (c) effects similar to those of wild type BiP were obtained with a deletion mutant devoid of chaperone activity. The specificity of the results was confirmed by parallel 45Ca experiments carried out in HeLa cells transfected with two other Ca2+-binding proteins, calreticulin and CaBP2(ERp72), only the first of which induced increases of Ca2+ capacity. We conclude that BiP has a dual function, in addition to its chaperone role it is a bona fide ER lumenal Ca2+ storage protein contributing, under resting cell conditions, to around 25% of the store, with a stoichiometry of 1–2 moles of calcium/mole of BiP. Studies carried out during the last decade have identified BiP as a ubiquitous lumenal resident protein of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Tg, thapsigargin; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ pump; PDI, protein disulfide isomerase; CNX, calnexin; CRT, calreticulin; PBS, phosphate-buffered saline; KRH, Krebs-Ringer-Hepes. 1The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Tg, thapsigargin; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ pump; PDI, protein disulfide isomerase; CNX, calnexin; CRT, calreticulin; PBS, phosphate-buffered saline; KRH, Krebs-Ringer-Hepes. (1Bole D.G. Hendershot L.M. Kearney J.F. J. Cell Biol. 1986; 102: 1558-1566Crossref PubMed Scopus (561) Google Scholar, 2Munro S. Pelham H.R. Cell. 1986; 46: 291-300Abstract Full Text PDF PubMed Scopus (1041) Google Scholar, 3Lee A.S. Trends. Biochem. Sci. 1987; 12: 20-23Abstract Full Text PDF Scopus (390) Google Scholar), playing a key role in the assistance of newly synthetized proteins for folding and acquisition of their correct tertiary and quaternary structure. Such a function, defined as a chaperone, implies the direct binding of BiP to the growing chains, with a stimulation of its ATPase activity (6Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3089) Google Scholar, 7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar, 8Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Abstract Full Text PDF PubMed Google Scholar, 9Bonifacino J.S. Lippincott-Schwartz J. Curr. Opin. Cell Biol. 1991; 3: 592-600Crossref PubMed Scopus (155) Google Scholar). When this task is not accomplished, misfolded proteins remain complexed to BiP (and to other chaperones) (7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar, 10Otsu M. Urade R. Kito M. Omura F. Kikuchi M. J. Biol. Chem. 1995; 270: 14958-14961Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) to be ultimately disposed of by nonlysosomal proteolytic process(es) (quality control) (7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar, 9Bonifacino J.S. Lippincott-Schwartz J. Curr. Opin. Cell Biol. 1991; 3: 592-600Crossref PubMed Scopus (155) Google Scholar, 10Otsu M. Urade R. Kito M. Omura F. Kikuchi M. J. Biol. Chem. 1995; 270: 14958-14961Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Werner E.D. Brodsky J.L. Mc Cracken A.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (386) Google Scholar). These fundamental activities are carried out by BiP working in the peculiar environment of the ER lumen (12Gaut J.R. Hendershot L.M. Curr. Opin. Cell Biol. 1993; 5: 589-595Crossref PubMed Scopus (116) Google Scholar) characterized, among other properties, by a high (mM) concentration of free Ca2+ ([Ca2+] er ) (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar, 14Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus (263) Google Scholar). Such a property of the environment appears to be of importance for BiP function. Indeed, previous studies carried out with both recombinant BiP protein and BiP purified from cells have shown that the chaperone ATPase activity is altered by Ca2+, and BiP association with proteins in vitro is stabilized by Ca2+ (8Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Abstract Full Text PDF PubMed Google Scholar,15Kassenbrock C.K. Kelly R.B. EMBO J. 1989; 8: 1461-1467Crossref PubMed Scopus (130) Google Scholar, 16Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 12691-12698Abstract Full Text PDF PubMed Google Scholar). Moreover, major changes of Ca2+ homeostasis, such as those induced by prolonged applications of Ca2+ionophores, have been shown to induce major disturbances in the cells, including inappropriate release and secretion of BiP-associated proteins (17Suzuki C.K. Bonifacino J.S. Lin A.Y. Davis M.M. Klausner R.D. J. Cell Biol. 1991; 114: 189-205Crossref PubMed Scopus (147) Google Scholar), alterations in the folding of ER proteins leading to the transcriptional up-regulation (3Lee A.S. Trends. Biochem. Sci. 1987; 12: 20-23Abstract Full Text PDF Scopus (390) Google Scholar, 18Dorner A.J. Wasley L.C. Raney P. Haugejorden S. Green M. Kaufman R.J. J. Biol. Chem. 1990; 265: 22029-22034Abstract Full Text PDF PubMed Google Scholar), and altered distribution of BiP and other chaperones (19Booth C. Koch G.L. Cell. 1989; 59: 729-737Abstract Full Text PDF PubMed Scopus (277) Google Scholar). So far, however, the possibility that BiP, in addition to its chaperone function, could also participate in the intralumenal storage of Ca2+, thus contributing to the accumulation of the rapidly exchanging pool of the cation, has never been investigated.In the present study we report that overexpression of exogenous hamster BiP in HeLa cells induces appreciable increases of the ER Ca2+ storage capacity as consistently revealed by two experimental approaches. In one, cells were cotransfected with the cDNA of the photoprotein aequorin that had been molecularly modified to assay [Ca2+] within two distinct intracellular compartments, either the cytosol or the mitochondria (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar,20Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-746Crossref PubMed Scopus (997) Google Scholar). In the other, the cells were loaded at the equilibrium with45Ca and release was induced by exposure to thapsigargin (Tg), a blocker of the sarcoplasmic/endoplasmic reticulum Ca2+ pump (SERCA). The latter treatment is known to induce the specific discharge of the ER compartment (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar, 21Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The Ca2+ storage effects observed with BiP overexpression were not due to its chaperone activity toward other proteins, since a BiP construct that lacks the protein binding domain produced similar results. Nor were these effects due to compensatory readjustments in the levels of other ER Ca2+-binding proteins, because the latter remained unchanged in the transfected cells. Moreover, parallel studies carried out in HeLa cells exposed to a stressful treatment with the glycosylation blocker drug, tunicamycin (22Dorner A.J. Wasley L.C. Kaufman R.J. EMBO J. 1992; 11: 1563-1571Crossref PubMed Scopus (297) Google Scholar), also led to an increase of the ER Ca2+ storage capacity, part of which is dependent on the stress-induced overexpression of BiP. We conclude that, in addition to its classical role of chaperone, BiP participates in a second function, the storage of Ca2+ within the ER lumen, and that the fluctuations of the protein induce adjustments of the cellular Ca2+ homeostasis.DISCUSSIONThe present results, obtained by two independent techniques, consistently reveal increased capacity of the rapidly exchanging ER Ca2+ store in the well characterized system of HeLa cells transiently transfected with either wild type BiP or its deletion mutant, 44KΔBiP. The two techniques employed should be considered complementary of each other. Cotransfection of the cytosol-targeted aequorin (28Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar) is advantageous not only because it provides the time course of the induced Ca2+ release responses but also because the origin of the Ca2+-triggered signal is restricted to cells expressing the photoprotein, which largely coincide with those overexpressing the cotransfected protein, i.e.the wild type or the 44KΔBiP. This property is important because in our transfected preparations only 35–50% of the cells expressed exogenous BiP on top of their endogenous complement of the protein. However, the aequorin signals depend not only on the Ca2+capacity, but also on other properties of the ER store (pumps, channels) as well as on homeostatic equilibria established within the cell. Therefore, they cannot be easily converted into quantitative data. 45Ca release, on the other hand, occurs from all the cells of the preparation (transfected and non transfected) which were first loaded at the equilibrium with the tracer. The advantage here is that, by using Tg, a blocker specific for the SERCAs, the results obtained are due to Ca2+ release only from the ER (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar).The significance of our findings deserves attention. As in all other overexpression experiments, results could in fact be due not (or not only) to the increased amounts of the investigated protein but (also) to indirect consequences induced by protein transfection. In the case of BiP, this concern appears particularly cogent in view of the chaperone function of this protein, with positive action on structure, and thus also on function, of other proteins, some of which contribute to ER Ca2+ homeostasis. However, the control results we have obtained do not seem to support these possibilities. The level of other proteins of the ER store, including CRT, which is known to be the major Ca2+ binder (34Michalak M. Milner R.E. Burns K. Opas M. Biochem. J. 1992; 285: 681-692Crossref PubMed Scopus (404) Google Scholar, 35Krause K.-H. Michalak M. Cell. 1997; 88: 1-11Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar), was in fact not significantly different in BiP-transfected and control cells. Changes of the ER store capacity due not to BiP but to BiP-induced changes of expression of other components appear therefore unlikely. Likewise, the consistence of the reported results obtained not only with BiP but also with its 44KΔBiP mutant, which is devoid of the peptide binding domain and thus of any chaperone function (23Hendershot L.M. Wei J.Y. Gaut J.R. Lawson B. Freiden P.J. Murti K.G. Mol. Biol. Cell. 1995; 6: 283-296Crossref PubMed Scopus (87) Google Scholar), excludes the involvement of a chaperone-induced improved function of the other Ca2+-binding proteins. It appears therefore that the effects observed are most likely due to direct, specific Ca2+ binding of the transfected protein. This conclusion is reinforced by the additional 45Ca data obtained using cells transfected not with BiP but with either CRT or CaBP2(ERp72). The results obtained on the one hand confirm that the contribution of CRT to the HeLa cell rapidly exchanging Ca2+ store amounts to ∼50% (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar) and on the other hand fail to show any detectable effect of CaBP2. In the cells overexpressing the latter protein, in fact, the apparent capacity of the ER in terms of Ca2+ storage was not increased but, if anything, slightly reduced.A role of BiP as one of the Ca2+ storage proteins of the ER lumen was proposed 10 years ago by Macer and Koch (40Macer D.R.J. Koch G.L.E. J. Cell Sci. 1988; 91: 61-70Crossref PubMed Google Scholar) who first observed a polyacrylamide gel electrophoresis band with an apparent molecular mass of ∼78 kDa positively labeled by 45Ca overlay. Subsequent studies by Van et al. (41Van P.N. Peter F. Soling H.D. J. Biol. Chem. 1989; 264: 17494-17501Abstract Full Text PDF PubMed Google Scholar), carried out by a more ample approach, revealed, however, that the45Ca-positive band of that size was not BiP but CaBP2(ERp72). In the meantime, evidence was accumulated demonstrating that for its chaperone function, in particular for the binding of proteins and ATPase activity (8Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Abstract Full Text PDF PubMed Google Scholar, 15Kassenbrock C.K. Kelly R.B. EMBO J. 1989; 8: 1461-1467Crossref PubMed Scopus (130) Google Scholar, 16Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 12691-12698Abstract Full Text PDF PubMed Google Scholar), BiP needs Ca2+ to be present in the medium. So far, however, the possibility that BiP contributes to the storage of the cation within the ER lumen, together with other proteins such as CRT, had never been envisaged. By the use of the aequorin approach in HeLa cells we demonstrate here that the contribution of the chaperone to the capacity of the typical rapidly exchanging Ca2+ pool is not insignificant. In fact, when investigated according to a two-stimulation protocol (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar), BiP overexpression was found to induce detectable increases of the Ca2+ release triggered by IP3 generation following receptor activation. This type of difference is not easy to evidentiate because it is extensively buffered by cytosolic and organelle mechanisms (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar, 42Pozzan T. Rizzuto R. Volpe P. Meldolesi J. Physiol. Rev. 1994; 74: 595-636Crossref PubMed Scopus (30) Google Scholar). Moreover, extension of the approach to mitochondria, carried out by studying cells expressing an aequorin construct addressed to those organelles (20Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-746Crossref PubMed Scopus (997) Google Scholar), revealed that the BiP contribution to the ER-segregated Ca2+ pool has a wide impact within the cell. In fact, similar to the cells overexpressing CRT, those transfected with BiP were able to show considerable [Ca2+] mt transient also in response to the second stimulation, when the responses of control cells were clearly reduced. In view of the importance of Ca2+ in mitochondrial physiology (36McCormack J.G. Halestrep A.P. Denton R.M. Physiol. Rev. 1990; 70: 391-495Crossref PubMed Scopus (1146) Google Scholar) these observations further document the importance of the BiP storage function in cell physiology.Interesting conclusions can be drawn also from the 45Ca results. The ∼25% increases of the Tg-induced release obtained from the transfected preparations, in which the overall BiP expression was twice as large as in the controls, suggest that the contribution of the endogenous chaperone corresponds to ∼25% as well. To reconstruct the whole ER segregated Ca2+ pool the BiP value should be added to that previously estimated for CRT, ∼50%, with the rest probably contributed by other components, not only proteins (lumenal and also of the membrane, such as CNX) but also nucleotides (42Pozzan T. Rizzuto R. Volpe P. Meldolesi J. Physiol. Rev. 1994; 74: 595-636Crossref PubMed Scopus (30) Google Scholar). Taking into account that in HeLa cells the concentration of BiP is considerable (∼1% of the protein, ∼5-fold higher than CRT), the above results suggest that the stoichiometry of ER lumenal Ca2+ binding, under resting conditions, is relatively low, i.e. between 1 and 2 moles of calcium/mole of protein. Since in the BiP sequence typical Ca2+ binding domains are not evident, it appears likely that the observed binding is due to the abundance of acidic amino acids, many of which arranged in doublets or triplets (43Ting J. Wooden S.K. Kriz R. Kelleher K. Kaufman R.J. Lee A.S. Gene (Amst.). 1987; 55: 147-152Crossref PubMed Scopus (52) Google Scholar, 44Lucero H.A. Lebeche D. Kaminer B. J. Biol. Chem. 1994; 269: 23112-23119Abstract Full Text PDF PubMed Google Scholar). Because of its role in Ca2+ accumulation and release, and by analogy with other storage proteins such as CRT and calsequestrin (42Pozzan T. Rizzuto R. Volpe P. Meldolesi J. Physiol. Rev. 1994; 74: 595-636Crossref PubMed Scopus (30) Google Scholar), this binding is expected to be of low affinity, although so far conclusive results with the purified protein have not been reported.A final comment concerns the results obtained with cells that, instead of being transfected, were exposed to tunicamycin to induce stress (27Wada I. Ou W.J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Abstract Full Text PDF PubMed Google Scholar). Under these conditions, overexpression of endogenous BiP, due to stimulated transcription, is a classical result (22Dorner A.J. Wasley L.C. Kaufman R.J. EMBO J. 1992; 11: 1563-1571Crossref PubMed Scopus (297) Google Scholar). Recently, a similar response has been reported for CRT (39Llewellyn D.H. Kendall F. Sheikh F.N. Campbell K. Biochem. J. 1996; 318: 550-560Crossref Scopus (51) Google Scholar). To our knowledge, however, neither the consequences of these events in terms of Ca2+ accumulation within intracellular stores nor the possibility of modulations in the ER Ca2+ capacity had so far ever been envisaged. Here we show that the capacity of ER stores, calculated on the basis of the 45Ca results, as described previously for CRT-overexpressing cells (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar), was considerably increased (68%). Based on the observed increases of both BiP and CRT, as revealed by Western blotting carried out in parallel, and on the ER resting Ca2+ storage levels established by 45Ca experiments with Tg, the two proteins were calculated to participate similarly to the overall increase, contributing ∼25 and 29%, respectively, above controls. Thus, the role of these chaperones during stress is not limited to the assistance and quality control of the ER proteins but includes also an increase of the Ca2+buffering within the endomembrane system, a process that could contribute to the protection of the cell from the Ca2+-dependent damage.In conclusion, the present results have added a new function to those already recognized for BiP, not only the classical chaperone, but also a Ca2+-binding protein playing an important role in the control of the ER lumenal Ca2+ homeostasis. Although typical not only of BiP but also of other ER proteins, such as CRT and CNX (7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar, 45Hebert D.N. Foellmer B. Helenius A. EMBO J. 1996; 15: 2961-2968Crossref PubMed Scopus (254) Google Scholar), the duality of function does not appear to be the rule since overexpression of another ER lumenal protein, CaBP2(ERp72), remained without appreciable consequences on cell Ca2+ homeostasis. This latter negative result with a protein that in vitro is known to bind Ca2+ (38Rupp K. Birnbach U. Lundstrom J. Van P.N. Soling H.D. J. Biol. Chem. 1994; 269: 2501-2507Abstract Full Text PDF PubMed Google Scholar,41Van P.N. Peter F. Soling H.D. J. Biol. Chem. 1989; 264: 17494-17501Abstract Full Text PDF PubMed Google Scholar) emphasizes the need for Ca2+ storage to be investigated also in living cells. At variance with CRT, where Ca2+binding and chaperoning are located in distinct domains (34Michalak M. Milner R.E. Burns K. Opas M. Biochem. J. 1992; 285: 681-692Crossref PubMed Scopus (404) Google Scholar, 35Krause K.-H. Michalak M. Cell. 1997; 88: 1-11Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar), in BiP the sites for the two functions might be intermixed, as suggested by the lack of obvious segregation in the polypeptide sequence (43Ting J. Wooden S.K. Kriz R. Kelleher K. Kaufman R.J. Lee A.S. Gene (Amst.). 1987; 55: 147-152Crossref PubMed Scopus (52) Google Scholar) and also by the Ca2+ regulation of the chaperone activity (8Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Abstract Full Text PDF PubMed Google Scholar,16Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 12691-12698Abstract Full Text PDF PubMed Google Scholar, 17Suzuki C.K. Bonifacino J.S. Lin A.Y. Davis M.M. Klausner R.D. J. Cell Biol. 1991; 114: 189-205Crossref PubMed Scopus (147) Google Scholar). The present studies have been carried out only in HeLa cells, which, however, are known to express, in their ER, levels of both BiP and CRT similar to those present in the ER of various other lines (7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar,34Michalak M. Milner R.E. Burns K. Opas M. Biochem. J. 1992; 285: 681-692Crossref PubMed Scopus (404) Google Scholar, 42Pozzan T. Rizzuto R. Volpe P. Meldolesi J. Physiol. Rev. 1994; 74: 595-636Crossref PubMed Scopus (30) Google Scholar). We conclude therefore that the two proteins together probably account for the bulk of the rapidly exchanging Ca2+ storage also in those other cells. The latter function, which is already known to play key roles in cell physiology, is shown here to be modulable, due to the changes of expression of the chaperones taking place during exposure to stressful stimuli. Studies carried out during the last decade have identified BiP as a ubiquitous lumenal resident protein of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Tg, thapsigargin; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ pump; PDI, protein disulfide isomerase; CNX, calnexin; CRT, calreticulin; PBS, phosphate-buffered saline; KRH, Krebs-Ringer-Hepes. 1The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Tg, thapsigargin; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ pump; PDI, protein disulfide isomerase; CNX, calnexin; CRT, calreticulin; PBS, phosphate-buffered saline; KRH, Krebs-Ringer-Hepes. (1Bole D.G. Hendershot L.M. Kearney J.F. J. Cell Biol. 1986; 102: 1558-1566Crossref PubMed Scopus (561) Google Scholar, 2Munro S. Pelham H.R. Cell. 1986; 46: 291-300Abstract Full Text PDF PubMed Scopus (1041) Google Scholar, 3Lee A.S. Trends. Biochem. Sci. 1987; 12: 20-23Abstract Full Text PDF Scopus (390) Google Scholar), playing a key role in the assistance of newly synthetized proteins for folding and acquisition of their correct tertiary and quaternary structure. Such a function, defined as a chaperone, implies the direct binding of BiP to the growing chains, with a stimulation of its ATPase activity (6Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3089) Google Scholar, 7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar, 8Gaut J.R. Hendershot L.M. J. Biol. Chem. 1993; 268: 7248-7255Abstract Full Text PDF PubMed Google Scholar, 9Bonifacino J.S. Lippincott-Schwartz J. Curr. Opin. Cell Biol. 1991; 3: 592-600Crossref PubMed Scopus (155) Google Scholar). When this task is not accomplished, misfolded proteins remain complexed to BiP (and to other chaperones) (7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar, 10Otsu M. Urade R. Kito M. Omura F. Kikuchi M. J. Biol. Chem. 1995; 270: 14958-14961Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) to be ultimately disposed of by nonlysosomal proteolytic process(es) (quality control) (7Brewer J.W. Hendershot L.M. Fink A. Goto Y. Molecular Chaperones in Proteins: Structure, Functions and Mode of Action. Marcel Decker, New York1997: 415-434Google Scholar, 9Bonifacino J.S. Lippincott-Schwartz J. Curr. Opin. Cell Biol. 1991; 3: 592-600Crossref PubMed Scopus (155) Google Scholar, 10Otsu M. Urade R. Kito M. Omura F. Kikuchi M. J. Biol. Chem. 1995; 270: 14958-14961Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Werner E.D. Brodsky J.L. Mc Cracken A.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (386) Google Scholar). These fundamental activities are carried out by BiP working in the peculiar environment of the ER lumen (12Gaut J.R. Hendershot L.M. Curr. Opin. Cell Biol. 1993; 5: 589-595Crossref PubMed Scopus (116) Google Scholar) characterized, among other properties, by a high (mM) concentration of free Ca2+ ([Ca2+] er ) (13Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (168) Google Scholar, 14Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus