SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091276136> ?p ?o ?g. }

Showing items 1 to 100 of ±116 with 100 items per page.

W2091276136 endingPage "37956" @default.
W2091276136 startingPage "37951" @default.
W2091276136 abstract "Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 °C. This strain supports the growth of phages T4 and T5 but not phage λ and produces light at 28 °C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 °C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is importantin vivo in a manner that depends on the physiological conditions and is protein substrate specific. Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 °C. This strain supports the growth of phages T4 and T5 but not phage λ and produces light at 28 °C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 °C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is importantin vivo in a manner that depends on the physiological conditions and is protein substrate specific. polymerase chain reaction plaque-forming units The Escherichia coli GroE system facilitates protein folding in vivo and in vitro in an ATP-dependent manner (for recent reviews see, for example, Refs. 1Sigler P.B. Xu Z. Rye H.S. Burston S.G. Fenton W.A. Horwich A.L. Annu. Rev. Biochem. 1998; 67: 581-608Crossref PubMed Scopus (473) Google Scholar, 2Ellis R.J. Hartl F.-U. Curr. Opin. Struct. Biol. 1999; 9: 102-110Crossref PubMed Scopus (267) Google Scholar, 3Ranson N.A. White H.E. Saibil H.R. Biochem. J. 1998; 333: 233-242Crossref PubMed Scopus (165) Google Scholar, 4Horovitz A. Curr. Opin. Struct. Biol. 1998; 8: 93-100Crossref PubMed Scopus (54) Google Scholar). It is composed of GroEL, an oligomer of 14 identical subunits that form two heptameric rings, stacked back-to-back, with 7-fold symmetry and a cavity at each end (5Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar), and its helper-protein GroES which is a seven-membered ring of identical subunits (6Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (397) Google Scholar). GroEL has 14 ATP-binding sites and a weak K+-dependent (7Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (310) Google Scholar) ATPase activity. It undergoes ATP-induced conformational changes (8Roseman A.M. Chen S. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) that are reflected in binding of ATP with intra-ring positive cooperativity (9Gray T.E. Fersht A.R. FEBS Lett. 1991; 292: 254-258Crossref PubMed Scopus (165) Google Scholar, 10Bochkareva E.S. Lissin N.M. Flynn G.C. Rothman J.E. Girshovich A.S. J. Biol. Chem. 1992; 267: 6796-6800Abstract Full Text PDF PubMed Google Scholar, 11Jackson G.S. Staniforth R.A. Halsall D.J. Atkinson T. Holbrook J.J. Clarke A.R. Burston S.G. Biochemistry. 1993; 32: 2554-2563Crossref PubMed Scopus (236) Google Scholar) and inter-ring negative cooperativity (12Yifrach O. Horovitz A. J. Mol. Biol. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar, 13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar).Coupling between protein folding and allostery in the GroE system has recently been demonstrated in vitro (14Yifrach O. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1521-1524Crossref PubMed Scopus (55) Google Scholar). The importance of the allosteric properties of GroEL for its function in vivoremains, however, unclear. It has been questioned due to (i) the fact that the allosteric transitions of GroEL take place in vitroat subphysiological (micromolar) concentrations of ATP (13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar), and (ii) the finding that the apical domain of GroEL, which is devoid of ATPase activity, is active in vivo (15Chatellier J. Hill F. Lund P.A. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9861-9866Crossref PubMed Scopus (63) Google Scholar). More recently, it has been demonstrated that the oligomeric structure of GroEL is required for biological activity because of the need for an intact cavity (16Wang J.D. Michelitsch M.D. Weissman J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12163-12168Crossref PubMed Scopus (56) Google Scholar, 17Weber F. Keppel F. Georgopoulos C. Hayer-Hartl M.K. Hartl F.-U. Nat. Struct. Biol. 1998; 5: 977-985Crossref PubMed Scopus (65) Google Scholar). Evidence for the importance of the oligomeric structure of GroEL for activity in vivo owing to the requirement for proper allosteric communication within and between rings has, however, not been reported. We decided to begin addressing this issue by generatingE. coli strains that express only plasmid-derived GroEL which is either wild type (as a control) or mutant with modified allosteric properties. The following GroEL mutants with different altered allosteric properties were chosen for analysis as follows: (i) GroEL(K4E) with disrupted inter-subunit contacts (18Horovitz A. Bochkareva E.S. Girshovich A.S. J. Biol. Chem. 1993; 268: 9957-9959Abstract Full Text PDF PubMed Google Scholar, 19White Z.W. Fisher K.E. Eisenstein E. J. Biol. Chem. 1995; 270: 20404-20409Crossref PubMed Scopus (31) Google Scholar); (ii) GroEL(R13G, A126V) with intact positive cooperativity and disrupted negative cooperativity (20Aharoni A. Horovitz A. J. Mol. Biol. 1996; 258: 732-735Crossref PubMed Scopus (45) Google Scholar); (iii) GroEL(R197A) with strongly diminished positive cooperativity and weakened negative cooperativity (12Yifrach O. Horovitz A. J. Mol. Biol. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar); (iv) GroEL(E409A, R501A) with increased positive and slightly weakened negative cooperativity (21Aharoni A. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1698-1702Crossref PubMed Scopus (19) Google Scholar), and (v) GroEL(R501A) with weakened positive cooperativity and disrupted negative cooperativity (21Aharoni A. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1698-1702Crossref PubMed Scopus (19) Google Scholar). To date, there have been very few studies on the in vivo consequences of mutations in GroEL known to modify its properties in vitro (see, for example, Ref. 22Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar). Herein, we show that such mutations affect the function of GroEL in vivo (in cells that lack background chromosomal wild-type GroEL) and demonstrate that the effects depend on the physiological conditions and are protein substrate-specific.DISCUSSIONBacteriophage P1 transduction was used to generate E. coli TG1 strains that express only plasmid-derived wild-type GroEL or mutants with various modified and well characterized allosteric properties. The effects of these mutations on the function of GroELin vivo were studied using the following two assays: (i) propagation of phages λ, T4, and T5 and (ii) the bioluminescence of cells containing the full lux operon of V. fischeri. The GroE system was first identified by genetic studies of bacteriophage growth (30Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (197) Google Scholar). Bacteriophages λ (31Sternberg N. J. Mol. Biol. 1973; 76: 25-44Crossref PubMed Scopus (100) Google Scholar, 32Georgopoulos C.P. Hendrix R.W. Casjens S.R. Kaiser A.D. J. Mol. Biol. 1973; 76: 45-60Crossref PubMed Scopus (272) Google Scholar) and T5 (33Zweig M. Cummings D.J. J. Mol. Biol. 1973; 80: 505-518Crossref PubMed Scopus (62) Google Scholar) employ the host GroE system for the folding of their own proteins. Bacteriophage T4 uses host GroEL but its own co-chaperonin, Gp31, for the folding of its major capsid protein, Gp23 (34Laemmli U.K. Beguin F. Gujer-Kellenberger G. J. Mol. Biol. 1970; 47: 69-85Crossref PubMed Scopus (281) Google Scholar, 35van der Vies S.M. Gatenby A.A. Georgopoulos C. Nature. 1994; 368: 654-656Crossref PubMed Scopus (89) Google Scholar). Luminescence inV. fischeri cells and in E. coli cells that contain the lux genes requires the product of theluxR gene which activates transcription of thelux operon upon binding to an autoinducer (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar). The GroE system is believed to facilitate the folding in vivo of the LuxR protein (36Dolan K.M. Greenberg E.P. J. Bacteriol. 1992; 174: 5132-5135Crossref PubMed Google Scholar, 37Adar Y.Y. Ulitzur S. J. Biolumin. Chemilumin. 1993; 8: 261-266Crossref PubMed Scopus (32) Google Scholar) and possibly also the luciferase α (LuxA) and β (LuxB) subunits (38Fedorov A.N. Baldwin T.O. J. Mol. Biol. 1997; 268: 712-723Crossref PubMed Scopus (29) Google Scholar). The lux operon also containsluxC, luxD, and luxE which code for enzymes required for synthesis of the long chain aldehyde luciferase substrate, luxI which codes for an enzyme involved in autoinducer synthesis and luxG which codes for a protein with unknown function (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar).The functional consequences in vivo of mutations that alter the allosteric properties of GroEL are found in this study to be protein substrate-specific. Cells containing GroEL(R197A), for example, grow poorly at 37 (not shown) and 42 °C (Fig. 1) and do not support growth of phage λ but do support growth of phages T4 and T5 (Fig. 2) and the folding of the lux operon gene products and other proteins that may be required for light production (Figs. 3 and 4). Cells containing GroEL(R501A) or GroEL(R13G, A126V), on the other hand, support the growth of phages λ, T4, and T5 (not shown) but not the folding at 28 °C of one or more of the proteins required for bioluminescence (Figs. 3 and 4). The need for specific allosteric properties in GroEL therefore depends on the nature of the protein substrates.The requirement for the GroE system for folding in vivo can be circumvented by changing conditions in a substrate-specific manner. All the TG1ΔEL/pBADEL strains examined in this study produce light at 20 °C (Fig. 4 B), thus suggesting that unassisted folding of the proteins required for bioluminescence is more efficient at this temperature as, for example, observed in the case of ribulose-bisphosphate carboxylase/oxygenase (7Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (310) Google Scholar). All the TG1ΔEL/pBADEL strains examined in this study also produce light at 28 °C when inducer is added (not shown), perhaps because the active conformation of LuxR is stabilized in its presence (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar). Light production by cells containing GroEL(R501A) or GroEL(R13G, A126V) at 28 °C is, however, very low in the presence of inducer. Interestingly, the TG1ΔEL/pBADEL(R197A) strain which grows poorly at 37 and 42 °C is the only one that produces relatively more light at a low cell density, perhaps because folding of proteins which inhibit luminescence, such as LexA (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar), is not facilitated by GroEL(R197A).The effects in vivo of mutations in GroEL are also found to depend on its level of expression. If the GroEL mutant has low affinity for unfolded protein substrates then increasing its concentration may reduce the effect of the mutation in vivo. This may explain why the TG1ΔEL/pBADEL(R197A) strain is not viable at 37 °C when the concentration of arabinose is less than 0.002% (Fig. 2). If, however, the GroEL mutant binds unfolded proteins but does not release them (i.e. it is a “trap” mutant), then lowering its concentration may diminish the effect of the mutation. For example, all the TG1ΔEL/pBADEL strains produce less light at 28 °C in the presence of 0.2% arabinose than in the presence of 0.02% arabinose (not shown). Changes in the expression level of GroEL also alter the GroEL/GroES ratio in the cell which, although physiologically important, does not affect the conclusions in this study since the amount of GroEL in the different strains was determined to be the same (not shown).An understanding of how the mutations in GroEL affect its functionin vivo requires identification of the relevant substrate protein(s) whose misfolding leads to the observed phenotype and establishing the mechanism by which the mutation causes the misfolding. Here, we concentrated on trying to understand the reasons for differences in bioluminescence of the different TG1ΔEL/pBADEL strains containing the lux operon. We initially focused on LuxR as the substrate of GroEL because of reports in the literature that GroE facilitates the folding in vivo of the LuxR protein (36Dolan K.M. Greenberg E.P. J. Bacteriol. 1992; 174: 5132-5135Crossref PubMed Google Scholar,37Adar Y.Y. Ulitzur S. J. Biolumin. Chemilumin. 1993; 8: 261-266Crossref PubMed Scopus (32) Google Scholar). GroEL was found to bind denatured LuxR and release it in an ATP- and GroES-dependent manner, but no differences in binding or release of LuxR by the different mutants were observed (not shown) in agreement with the small effect of the autoinducer on light production by the TG1ΔEL/pBADEL(R501A) and TG1ΔEL/pBADEL(R13G, A126V) strains. Next we analyzed GroE-assisted reactivation of denatured α and β luciferase subunits. The GroE system was previously shown to facilitate the in vitro folding of bacterial luciferase from Vibrio harveyi (38Fedorov A.N. Baldwin T.O. J. Mol. Biol. 1997; 268: 712-723Crossref PubMed Scopus (29) Google Scholar). The extent of reactivation of α and β luciferase subunits by wild-type GroEL and the various mutants, in the presence of GroES and ATP, was found to be similar except in the case of GroEL(R13G, A126V) where the yield was about 50% that of the others (Fig. 5 C). GroEL(R13G, A126V) lacks negative cooperativity between rings but has intact intra-ring positive cooperativity and ak cat of ATP hydrolysis similar to that of wild-type GroEL (20Aharoni A. Horovitz A. J. Mol. Biol. 1996; 258: 732-735Crossref PubMed Scopus (45) Google Scholar). This mutant was also found to differ from wild-type GroEL and the other mutants in being able to release bound luciferase in the presence of ATP alone (Fig. 5 B). It was recently shown that the ATP-bound conformation of GroEL(A126V) is similar to the GroES-bound conformation of wild-type GroEL (39Llorca O. Perez-Perez J. Carrascosa J.L. Galan A. Muga A. Valpuesta J.M. J. Biol. Chem. 1997; 272: 32925-32932Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) thus explaining why ATP by itself can trigger release of substrates bound to GroEL(R13G, A126V). ATP-triggered release of luciferase subunits in a conformation not yet committed to fold may contribute to the lower yield of folding in the presence of GroEL(R13G, A126V) relative to wild-type GroEL. The GroEL(A126V) mutant was also found to form symmetric 2:1 GroES-GroEL complexes (39Llorca O. Perez-Perez J. Carrascosa J.L. Galan A. Muga A. Valpuesta J.M. J. Biol. Chem. 1997; 272: 32925-32932Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) in which substrate-binding sites are blocked. These properties of GroEL(A126V) may explain why cells containing GroEL(R13G, A126V) do not produce light at 28 °C. We do not yet know which proteins required for bioluminescence fail to fold in the presence of GroEL(R501A) and also which proteins involved in cell growth and λ phage propagation fail to fold in the presence of GroEL(R197A) at 25 and 37 °C.Our results suggest that mutations that perturb specific steps in the reaction cycle of GroEL are likely to be relatively more damaging. For example, cells containing only GroEL(D398A), which is defective in ATP hydrolysis, are not viable (not shown), whereas cells containing GroEL(K4E), which tends to dissociate into monomers (18Horovitz A. Bochkareva E.S. Girshovich A.S. J. Biol. Chem. 1993; 268: 9957-9959Abstract Full Text PDF PubMed Google Scholar, 19White Z.W. Fisher K.E. Eisenstein E. J. Biol. Chem. 1995; 270: 20404-20409Crossref PubMed Scopus (31) Google Scholar), exhibit a normal phenotype. This phenotype may be due, in part, to assembly into oligomeric structures in the presence of physiological concentrations of ATP (data not shown). GroEL(K4E) and also minichaperones (15Chatellier J. Hill F. Lund P.A. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9861-9866Crossref PubMed Scopus (63) Google Scholar) may retain some chaperoning function owing to mass action without trapping substrate proteins.In summary, our results indicate that allosteric communication in GroEL is important for the in vivo folding of a subset of substrates under certain physiological conditions. The poor growth of TG1ΔEL/pBADEL(R197A) at 42 °C and the formation of inclusion bodies in these cells (not shown) suggests that intact positive cooperativity may be of particular importance under stress conditions. Disrupted positive cooperativity reduces the shift in equilibrium under stress conditions toward the protein acceptor state of GroEL. Hartl and co-workers (40Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.-U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (432) Google Scholar) recently identified a set of in vivosubstrates of GroEL. Our results suggest that a “universal” set of protein substrates does not exist and that the set of substrates that interact with GroEL in vivo depends on the physiological conditions. The Escherichia coli GroE system facilitates protein folding in vivo and in vitro in an ATP-dependent manner (for recent reviews see, for example, Refs. 1Sigler P.B. Xu Z. Rye H.S. Burston S.G. Fenton W.A. Horwich A.L. Annu. Rev. Biochem. 1998; 67: 581-608Crossref PubMed Scopus (473) Google Scholar, 2Ellis R.J. Hartl F.-U. Curr. Opin. Struct. Biol. 1999; 9: 102-110Crossref PubMed Scopus (267) Google Scholar, 3Ranson N.A. White H.E. Saibil H.R. Biochem. J. 1998; 333: 233-242Crossref PubMed Scopus (165) Google Scholar, 4Horovitz A. Curr. Opin. Struct. Biol. 1998; 8: 93-100Crossref PubMed Scopus (54) Google Scholar). It is composed of GroEL, an oligomer of 14 identical subunits that form two heptameric rings, stacked back-to-back, with 7-fold symmetry and a cavity at each end (5Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar), and its helper-protein GroES which is a seven-membered ring of identical subunits (6Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (397) Google Scholar). GroEL has 14 ATP-binding sites and a weak K+-dependent (7Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (310) Google Scholar) ATPase activity. It undergoes ATP-induced conformational changes (8Roseman A.M. Chen S. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) that are reflected in binding of ATP with intra-ring positive cooperativity (9Gray T.E. Fersht A.R. FEBS Lett. 1991; 292: 254-258Crossref PubMed Scopus (165) Google Scholar, 10Bochkareva E.S. Lissin N.M. Flynn G.C. Rothman J.E. Girshovich A.S. J. Biol. Chem. 1992; 267: 6796-6800Abstract Full Text PDF PubMed Google Scholar, 11Jackson G.S. Staniforth R.A. Halsall D.J. Atkinson T. Holbrook J.J. Clarke A.R. Burston S.G. Biochemistry. 1993; 32: 2554-2563Crossref PubMed Scopus (236) Google Scholar) and inter-ring negative cooperativity (12Yifrach O. Horovitz A. J. Mol. Biol. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar, 13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar). Coupling between protein folding and allostery in the GroE system has recently been demonstrated in vitro (14Yifrach O. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1521-1524Crossref PubMed Scopus (55) Google Scholar). The importance of the allosteric properties of GroEL for its function in vivoremains, however, unclear. It has been questioned due to (i) the fact that the allosteric transitions of GroEL take place in vitroat subphysiological (micromolar) concentrations of ATP (13Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar), and (ii) the finding that the apical domain of GroEL, which is devoid of ATPase activity, is active in vivo (15Chatellier J. Hill F. Lund P.A. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9861-9866Crossref PubMed Scopus (63) Google Scholar). More recently, it has been demonstrated that the oligomeric structure of GroEL is required for biological activity because of the need for an intact cavity (16Wang J.D. Michelitsch M.D. Weissman J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12163-12168Crossref PubMed Scopus (56) Google Scholar, 17Weber F. Keppel F. Georgopoulos C. Hayer-Hartl M.K. Hartl F.-U. Nat. Struct. Biol. 1998; 5: 977-985Crossref PubMed Scopus (65) Google Scholar). Evidence for the importance of the oligomeric structure of GroEL for activity in vivo owing to the requirement for proper allosteric communication within and between rings has, however, not been reported. We decided to begin addressing this issue by generatingE. coli strains that express only plasmid-derived GroEL which is either wild type (as a control) or mutant with modified allosteric properties. The following GroEL mutants with different altered allosteric properties were chosen for analysis as follows: (i) GroEL(K4E) with disrupted inter-subunit contacts (18Horovitz A. Bochkareva E.S. Girshovich A.S. J. Biol. Chem. 1993; 268: 9957-9959Abstract Full Text PDF PubMed Google Scholar, 19White Z.W. Fisher K.E. Eisenstein E. J. Biol. Chem. 1995; 270: 20404-20409Crossref PubMed Scopus (31) Google Scholar); (ii) GroEL(R13G, A126V) with intact positive cooperativity and disrupted negative cooperativity (20Aharoni A. Horovitz A. J. Mol. Biol. 1996; 258: 732-735Crossref PubMed Scopus (45) Google Scholar); (iii) GroEL(R197A) with strongly diminished positive cooperativity and weakened negative cooperativity (12Yifrach O. Horovitz A. J. Mol. Biol. 1994; 243: 397-401Crossref PubMed Scopus (97) Google Scholar); (iv) GroEL(E409A, R501A) with increased positive and slightly weakened negative cooperativity (21Aharoni A. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1698-1702Crossref PubMed Scopus (19) Google Scholar), and (v) GroEL(R501A) with weakened positive cooperativity and disrupted negative cooperativity (21Aharoni A. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1698-1702Crossref PubMed Scopus (19) Google Scholar). To date, there have been very few studies on the in vivo consequences of mutations in GroEL known to modify its properties in vitro (see, for example, Ref. 22Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar). Herein, we show that such mutations affect the function of GroEL in vivo (in cells that lack background chromosomal wild-type GroEL) and demonstrate that the effects depend on the physiological conditions and are protein substrate-specific. DISCUSSIONBacteriophage P1 transduction was used to generate E. coli TG1 strains that express only plasmid-derived wild-type GroEL or mutants with various modified and well characterized allosteric properties. The effects of these mutations on the function of GroELin vivo were studied using the following two assays: (i) propagation of phages λ, T4, and T5 and (ii) the bioluminescence of cells containing the full lux operon of V. fischeri. The GroE system was first identified by genetic studies of bacteriophage growth (30Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (197) Google Scholar). Bacteriophages λ (31Sternberg N. J. Mol. Biol. 1973; 76: 25-44Crossref PubMed Scopus (100) Google Scholar, 32Georgopoulos C.P. Hendrix R.W. Casjens S.R. Kaiser A.D. J. Mol. Biol. 1973; 76: 45-60Crossref PubMed Scopus (272) Google Scholar) and T5 (33Zweig M. Cummings D.J. J. Mol. Biol. 1973; 80: 505-518Crossref PubMed Scopus (62) Google Scholar) employ the host GroE system for the folding of their own proteins. Bacteriophage T4 uses host GroEL but its own co-chaperonin, Gp31, for the folding of its major capsid protein, Gp23 (34Laemmli U.K. Beguin F. Gujer-Kellenberger G. J. Mol. Biol. 1970; 47: 69-85Crossref PubMed Scopus (281) Google Scholar, 35van der Vies S.M. Gatenby A.A. Georgopoulos C. Nature. 1994; 368: 654-656Crossref PubMed Scopus (89) Google Scholar). Luminescence inV. fischeri cells and in E. coli cells that contain the lux genes requires the product of theluxR gene which activates transcription of thelux operon upon binding to an autoinducer (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar). The GroE system is believed to facilitate the folding in vivo of the LuxR protein (36Dolan K.M. Greenberg E.P. J. Bacteriol. 1992; 174: 5132-5135Crossref PubMed Google Scholar, 37Adar Y.Y. Ulitzur S. J. Biolumin. Chemilumin. 1993; 8: 261-266Crossref PubMed Scopus (32) Google Scholar) and possibly also the luciferase α (LuxA) and β (LuxB) subunits (38Fedorov A.N. Baldwin T.O. J. Mol. Biol. 1997; 268: 712-723Crossref PubMed Scopus (29) Google Scholar). The lux operon also containsluxC, luxD, and luxE which code for enzymes required for synthesis of the long chain aldehyde luciferase substrate, luxI which codes for an enzyme involved in autoinducer synthesis and luxG which codes for a protein with unknown function (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar).The functional consequences in vivo of mutations that alter the allosteric properties of GroEL are found in this study to be protein substrate-specific. Cells containing GroEL(R197A), for example, grow poorly at 37 (not shown) and 42 °C (Fig. 1) and do not support growth of phage λ but do support growth of phages T4 and T5 (Fig. 2) and the folding of the lux operon gene products and other proteins that may be required for light production (Figs. 3 and 4). Cells containing GroEL(R501A) or GroEL(R13G, A126V), on the other hand, support the growth of phages λ, T4, and T5 (not shown) but not the folding at 28 °C of one or more of the proteins required for bioluminescence (Figs. 3 and 4). The need for specific allosteric properties in GroEL therefore depends on the nature of the protein substrates.The requirement for the GroE system for folding in vivo can be circumvented by changing conditions in a substrate-specific manner. All the TG1ΔEL/pBADEL strains examined in this study produce light at 20 °C (Fig. 4 B), thus suggesting that unassisted folding of the proteins required for bioluminescence is more efficient at this temperature as, for example, observed in the case of ribulose-bisphosphate carboxylase/oxygenase (7Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (310) Google Scholar). All the TG1ΔEL/pBADEL strains examined in this study also produce light at 28 °C when inducer is added (not shown), perhaps because the active conformation of LuxR is stabilized in its presence (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar). Light production by cells containing GroEL(R501A) or GroEL(R13G, A126V) at 28 °C is, however, very low in the presence of inducer. Interestingly, the TG1ΔEL/pBADEL(R197A) strain which grows poorly at 37 and 42 °C is the only one that produces relatively more light at a low cell density, perhaps because folding of proteins which inhibit luminescence, such as LexA (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar), is not facilitated by GroEL(R197A).The effects in vivo of mutations in GroEL are also found to depend on its level of expression. If the GroEL mutant has low affinity for unfolded protein substrates then increasing its concentration may reduce the effect of the mutation in vivo. This may explain why the TG1ΔEL/pBADEL(R197A) strain is not viable at 37 °C when the concentration of arabinose is less than 0.002% (Fig. 2). If, however, the GroEL mutant binds unfolded proteins but does not release them (i.e. it is a “trap” mutant), then lowering its concentration may diminish the effect of the mutation. For example, all the TG1ΔEL/pBADEL strains produce less light at 28 °C in the presence of 0.2% arabinose than in the presence of 0.02% arabinose (not shown). Changes in the expression level of GroEL also alter the GroEL/GroES ratio in the cell which, although physiologically important, does not affect the conclusions in this study since the amount of GroEL in the different strains was determined to be the same (not shown).An understanding of how the mutations in GroEL affect its functionin vivo requires identification of the relevant substrate protein(s) whose misfolding leads to the observed phenotype and establishing the mechanism by which the mutation causes the misfolding. Here, we concentrated on trying to understand the reasons for differences in bioluminescence of the different TG1ΔEL/pBADEL strains containing the lux operon. We initially focused on LuxR as the substrate of GroEL because of reports in the literature that GroE facilitates the folding in vivo of the LuxR protein (36Dolan K.M. Greenberg E.P. J. Bacteriol. 1992; 174: 5132-5135Crossref PubMed Google Scholar,37Adar Y.Y. Ulitzur S. J. Biolumin. Chemilumin. 1993; 8: 261-266Crossref PubMed Scopus (32) Google Scholar). GroEL was found to bind denatured LuxR and release it in an ATP- and GroES-dependent manner, but no differences in binding or release of LuxR by the different mutants were observed (not shown) in agreement with the small effect of the autoinducer on light production by the TG1ΔEL/pBADEL(R501A) and TG1ΔEL/pBADEL(R13G, A126V) strains. Next we analyzed GroE-assisted reactivation of denatured α and β luciferase subunits. The GroE system was previously shown to facilitate the in vitro folding of bacterial luciferase from Vibrio harveyi (38Fedorov A.N. Baldwin T.O. J. Mol. Biol. 1997; 268: 712-723Crossref PubMed Scopus (29) Google Scholar). The extent of reactivation of α and β luciferase subunits by wild-type GroEL and the various mutants, in the presence of GroES and ATP, was found to be similar except in the case of GroEL(R13G, A126V) where the yield was about 50% that of the others (Fig. 5 C). GroEL(R13G, A126V) lacks negative cooperativity between rings but has intact intra-ring positive cooperativity and ak cat of ATP hydrolysis similar to that of wild-type GroEL (20Aharoni A. Horovitz A. J. Mol. Biol. 1996; 258: 732-735Crossref PubMed Scopus (45) Google Scholar). This mutant was also found to differ from wild-type GroEL and the other mutants in being able to release bound luciferase in the presence of ATP alone (Fig. 5 B). It was recently shown that the ATP-bound conformation of GroEL(A126V) is similar to the GroES-bound conformation of wild-type GroEL (39Llorca O. Perez-Perez J. Carrascosa J.L. Galan A. Muga A. Valpuesta J.M. J. Biol. Chem. 1997; 272: 32925-32932Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) thus explaining why ATP by itself can trigger release of substrates bound to GroEL(R13G, A126V). ATP-triggered release of luciferase subunits in a conformation not yet committed to fold may contribute to the lower yield of folding in the presence of GroEL(R13G, A126V) relative to wild-type GroEL. The GroEL(A126V) mutant was also found to form symmetric 2:1 GroES-GroEL complexes (39Llorca O. Perez-Perez J. Carrascosa J.L. Galan A. Muga A. Valpuesta J.M. J. Biol. Chem. 1997; 272: 32925-32932Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) in which substrate-binding sites are blocked. These properties of GroEL(A126V) may explain why cells containing GroEL(R13G, A126V) do not produce light at 28 °C. We do not yet know which proteins required for bioluminescence fail to fold in the presence of GroEL(R501A) and also which proteins involved in cell growth and λ phage propagation fail to fold in the presence of GroEL(R197A) at 25 and 37 °C.Our results suggest that mutations that perturb specific steps in the reaction cycle of GroEL are likely to be relatively more damaging. For example, cells containing only GroEL(D398A), which is defective in ATP hydrolysis, are not viable (not shown), whereas cells containing GroEL(K4E), which tends to dissociate into monomers (18Horovitz A. Bochkareva E.S. Girshovich A.S. J. Biol. Chem. 1993; 268: 9957-9959Abstract Full Text PDF PubMed Google Scholar, 19White Z.W. Fisher K.E. Eisenstein E. J. Biol. Chem. 1995; 270: 20404-20409Crossref PubMed Scopus (31) Google Scholar), exhibit a normal phenotype. This phenotype may be due, in part, to assembly into oligomeric structures in the presence of physiological concentrations of ATP (data not shown). GroEL(K4E) and also minichaperones (15Chatellier J. Hill F. Lund P.A. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9861-9866Crossref PubMed Scopus (63) Google Scholar) may retain some chaperoning function owing to mass action without trapping substrate proteins.In summary, our results indicate that allosteric communication in GroEL is important for the in vivo folding of a subset of substrates under certain physiological conditions. The poor growth of TG1ΔEL/pBADEL(R197A) at 42 °C and the formation of inclusion bodies in these cells (not shown) suggests that intact positive cooperativity may be of particular importance under stress conditions. Disrupted positive cooperativity reduces the shift in equilibrium under stress conditions toward the protein acceptor state of GroEL. Hartl and co-workers (40Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.-U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (432) Google Scholar) recently identified a set of in vivosubstrates of GroEL. Our results suggest that a “universal” set of protein substrates does not exist and that the set of substrates that interact with GroEL in vivo depends on the physiological conditions. Bacteriophage P1 transduction was used to generate E. coli TG1 strains that express only plasmid-derived wild-type GroEL or mutants with various modified and well characterized allosteric properties. The effects of these mutations on the function of GroELin vivo were studied using the following two assays: (i) propagation of phages λ, T4, and T5 and (ii) the bioluminescence of cells containing the full lux operon of V. fischeri. The GroE system was first identified by genetic studies of bacteriophage growth (30Georgopoulos C. Trends Biochem. Sci. 1992; 17: 295-299Abstract Full Text PDF PubMed Scopus (197) Google Scholar). Bacteriophages λ (31Sternberg N. J. Mol. Biol. 1973; 76: 25-44Crossref PubMed Scopus (100) Google Scholar, 32Georgopoulos C.P. Hendrix R.W. Casjens S.R. Kaiser A.D. J. Mol. Biol. 1973; 76: 45-60Crossref PubMed Scopus (272) Google Scholar) and T5 (33Zweig M. Cummings D.J. J. Mol. Biol. 1973; 80: 505-518Crossref PubMed Scopus (62) Google Scholar) employ the host GroE system for the folding of their own proteins. Bacteriophage T4 uses host GroEL but its own co-chaperonin, Gp31, for the folding of its major capsid protein, Gp23 (34Laemmli U.K. Beguin F. Gujer-Kellenberger G. J. Mol. Biol. 1970; 47: 69-85Crossref PubMed Scopus (281) Google Scholar, 35van der Vies S.M. Gatenby A.A. Georgopoulos C. Nature. 1994; 368: 654-656Crossref PubMed Scopus (89) Google Scholar). Luminescence inV. fischeri cells and in E. coli cells that contain the lux genes requires the product of theluxR gene which activates transcription of thelux operon upon binding to an autoinducer (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar). The GroE system is believed to facilitate the folding in vivo of the LuxR protein (36Dolan K.M. Greenberg E.P. J. Bacteriol. 1992; 174: 5132-5135Crossref PubMed Google Scholar, 37Adar Y.Y. Ulitzur S. J. Biolumin. Chemilumin. 1993; 8: 261-266Crossref PubMed Scopus (32) Google Scholar) and possibly also the luciferase α (LuxA) and β (LuxB) subunits (38Fedorov A.N. Baldwin T.O. J. Mol. Biol. 1997; 268: 712-723Crossref PubMed Scopus (29) Google Scholar). The lux operon also containsluxC, luxD, and luxE which code for enzymes required for synthesis of the long chain aldehyde luciferase substrate, luxI which codes for an enzyme involved in autoinducer synthesis and luxG which codes for a protein with unknown function (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar). The functional consequences in vivo of mutations that alter the allosteric properties of GroEL are found in this study to be protein substrate-specific. Cells containing GroEL(R197A), for example, grow poorly at 37 (not shown) and 42 °C (Fig. 1) and do not support growth of phage λ but do support growth of phages T4 and T5 (Fig. 2) and the folding of the lux operon gene products and other proteins that may be required for light production (Figs. 3 and 4). Cells containing GroEL(R501A) or GroEL(R13G, A126V), on the other hand, support the growth of phages λ, T4, and T5 (not shown) but not the folding at 28 °C of one or more of the proteins required for bioluminescence (Figs. 3 and 4). The need for specific allosteric properties in GroEL therefore depends on the nature of the protein substrates. The requirement for the GroE system for folding in vivo can be circumvented by changing conditions in a substrate-specific manner. All the TG1ΔEL/pBADEL strains examined in this study produce light at 20 °C (Fig. 4 B), thus suggesting that unassisted folding of the proteins required for bioluminescence is more efficient at this temperature as, for example, observed in the case of ribulose-bisphosphate carboxylase/oxygenase (7Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (310) Google Scholar). All the TG1ΔEL/pBADEL strains examined in this study also produce light at 28 °C when inducer is added (not shown), perhaps because the active conformation of LuxR is stabilized in its presence (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar). Light production by cells containing GroEL(R501A) or GroEL(R13G, A126V) at 28 °C is, however, very low in the presence of inducer. Interestingly, the TG1ΔEL/pBADEL(R197A) strain which grows poorly at 37 and 42 °C is the only one that produces relatively more light at a low cell density, perhaps because folding of proteins which inhibit luminescence, such as LexA (27Ulitzur S. Dunlap P.V. Photochem. Photobiol. 1995; 62: 625-632Crossref Scopus (44) Google Scholar), is not facilitated by GroEL(R197A). The effects in vivo of mutations in GroEL are also found to depend on its level of expression. If the GroEL mutant has low affinity for unfolded protein substrates then increasing its concentration may reduce the effect of the mutation in vivo. This may explain why the TG1ΔEL/pBADEL(R197A) strain is not viable at 37 °C when the concentration of arabinose is less than 0.002% (Fig. 2). If, however, the GroEL mutant binds unfolded proteins but does not release them (i.e. it is a “trap” mutant), then lowering its concentration may diminish the effect of the mutation. For example, all the TG1ΔEL/pBADEL strains produce less light at 28 °C in the presence of 0.2% arabinose than in the presence of 0.02% arabinose (not shown). Changes in the expression level of GroEL also alter the GroEL/GroES ratio in the cell which, although physiologically important, does not affect the conclusions in this study since the amount of GroEL in the different strains was determined to be the same (not shown). An understanding of how the mutations in GroEL affect its functionin vivo requires identification of the relevant substrate protein(s) whose misfolding leads to the observed phenotype and establishing the mechanism by which the mutation causes the misfolding. Here, we concentrated on trying to understand the reasons for differences in bioluminescence of the different TG1ΔEL/pBADEL strains containing the lux operon. We initially focused on LuxR as the substrate of GroEL because of reports in the literature that GroE facilitates the folding in vivo of the LuxR protein (36Dolan K.M. Greenberg E.P. J. Bacteriol. 1992; 174: 5132-5135Crossref PubMed Google Scholar,37Adar Y.Y. Ulitzur S. J. Biolumin. Chemilumin. 1993; 8: 261-266Crossref PubMed Scopus (32) Google Scholar). GroEL was found to bind denatured LuxR and release it in an ATP- and GroES-dependent manner, but no differences in binding or release of LuxR by the different mutants were observed (not shown) in agreement with the small effect of the autoinducer on light production by the TG1ΔEL/pBADEL(R501A) and TG1ΔEL/pBADEL(R13G, A126V) strains. Next we analyzed GroE-assisted reactivation of denatured α and β luciferase subunits. The GroE system was previously shown to facilitate the in vitro folding of bacterial luciferase from Vibrio harveyi (38Fedorov A.N. Baldwin T.O. J. Mol. Biol. 1997; 268: 712-723Crossref PubMed Scopus (29) Google Scholar). The extent of reactivation of α and β luciferase subunits by wild-type GroEL and the various mutants, in the presence of GroES and ATP, was found to be similar except in the case of GroEL(R13G, A126V) where the yield was about 50% that of the others (Fig. 5 C). GroEL(R13G, A126V) lacks negative cooperativity between rings but has intact intra-ring positive cooperativity and ak cat of ATP hydrolysis similar to that of wild-type GroEL (20Aharoni A. Horovitz A. J. Mol. Biol. 1996; 258: 732-735Crossref PubMed Scopus (45) Google Scholar). This mutant was also found to differ from wild-type GroEL and the other mutants in being able to release bound luciferase in the presence of ATP alone (Fig. 5 B). It was recently shown that the ATP-bound conformation of GroEL(A126V) is similar to the GroES-bound conformation of wild-type GroEL (39Llorca O. Perez-Perez J. Carrascosa J.L. Galan A. Muga A. Valpuesta J.M. J. Biol. Chem. 1997; 272: 32925-32932Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) thus explaining why ATP by itself can trigger release of substrates bound to GroEL(R13G, A126V). ATP-triggered release of luciferase subunits in a conformation not yet committed to fold may contribute to the lower yield of folding in the presence of GroEL(R13G, A126V) relative to wild-type GroEL. The GroEL(A126V) mutant was also found to form symmetric 2:1 GroES-GroEL complexes (39Llorca O. Perez-Perez J. Carrascosa J.L. Galan A. Muga A. Valpuesta J.M. J. Biol. Chem. 1997; 272: 32925-32932Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) in which substrate-binding sites are blocked. These properties of GroEL(A126V) may explain why cells containing GroEL(R13G, A126V) do not produce light at 28 °C. We do not yet know which proteins required for bioluminescence fail to fold in the presence of GroEL(R501A) and also which proteins involved in cell growth and λ phage propagation fail to fold in the presence of GroEL(R197A) at 25 and 37 °C. Our results suggest that mutations that perturb specific steps in the reaction cycle of GroEL are likely to be relatively more damaging. For example, cells containing only GroEL(D398A), which is defective in ATP hydrolysis, are not viable (not shown), whereas cells containing GroEL(K4E), which tends to dissociate into monomers (18Horovitz A. Bochkareva E.S. Girshovich A.S. J. Biol. Chem. 1993; 268: 9957-9959Abstract Full Text PDF PubMed Google Scholar, 19White Z.W. Fisher K.E. Eisenstein E. J. Biol. Chem. 1995; 270: 20404-20409Crossref PubMed Scopus (31) Google Scholar), exhibit a normal phenotype. This phenotype may be due, in part, to assembly into oligomeric structures in the presence of physiological concentrations of ATP (data not shown). GroEL(K4E) and also minichaperones (15Chatellier J. Hill F. Lund P.A. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9861-9866Crossref PubMed Scopus (63) Google Scholar) may retain some chaperoning function owing to mass action without trapping substrate proteins. In summary, our results indicate that allosteric communication in GroEL is important for the in vivo folding of a subset of substrates under certain physiological conditions. The poor growth of TG1ΔEL/pBADEL(R197A) at 42 °C and the formation of inclusion bodies in these cells (not shown) suggests that intact positive cooperativity may be of particular importance under stress conditions. Disrupted positive cooperativity reduces the shift in equilibrium under stress conditions toward the protein acceptor state of GroEL. Hartl and co-workers (40Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.-U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (432) Google Scholar) recently identified a set of in vivosubstrates of GroEL. Our results suggest that a “universal” set of protein substrates does not exist and that the set of substrates that interact with GroEL in vivo depends on the physiological conditions. We thank Dr. P. Lund for phage P1, the AI90/pBADEL strain, and helpful advice and Dr. S. van der Vies for phages λ, T4, and T5." @default.
W2091276136 created "2016-06-24" @default.
W2091276136 creator A5014486140 @default.
W2091276136 creator A5026193000 @default.
W2091276136 creator A5052416355 @default.
W2091276136 date "2000-12-01" @default.
W2091276136 modified "2023-09-30" @default.
W2091276136 title "In Vivo and in Vitro Function of GroEL Mutants with Impaired Allosteric Properties" @default.
W2091276136 cites W1480951920 @default.
W2091276136 cites W1486201417 @default.
W2091276136 cites W1518061853 @default.
W2091276136 cites W1533808701 @default.
W2091276136 cites W1563291525 @default.
W2091276136 cites W1589182547 @default.
W2091276136 cites W1892996896 @default.
W2091276136 cites W1968308190 @default.
W2091276136 cites W1974723963 @default.
W2091276136 cites W1975222498 @default.
W2091276136 cites W1989999739 @default.
W2091276136 cites W1993674986 @default.
W2091276136 cites W1995268731 @default.
W2091276136 cites W1996139069 @default.
W2091276136 cites W1999197280 @default.
W2091276136 cites W2000363955 @default.
W2091276136 cites W2002542088 @default.
W2091276136 cites W2006439901 @default.
W2091276136 cites W2017736821 @default.
W2091276136 cites W2018096567 @default.
W2091276136 cites W2021701576 @default.
W2091276136 cites W2022796050 @default.
W2091276136 cites W2027298000 @default.
W2091276136 cites W2028322882 @default.
W2091276136 cites W2032730244 @default.
W2091276136 cites W2035247586 @default.
W2091276136 cites W2042905595 @default.
W2091276136 cites W2044106262 @default.
W2091276136 cites W2047983557 @default.
W2091276136 cites W2055925084 @default.
W2091276136 cites W2065458144 @default.
W2091276136 cites W2080980515 @default.
W2091276136 cites W2083892680 @default.
W2091276136 cites W2132463414 @default.
W2091276136 cites W2142955330 @default.
W2091276136 cites W2144263933 @default.
W2091276136 cites W2154647343 @default.
W2091276136 cites W2160084442 @default.
W2091276136 cites W4229508230 @default.
W2091276136 doi "https://doi.org/10.1074/jbc.m007594200" @default.
W2091276136 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10973985" @default.
W2091276136 hasPublicationYear "2000" @default.
W2091276136 type Work @default.
W2091276136 sameAs 2091276136 @default.
W2091276136 citedByCount "13" @default.
W2091276136 countsByYear W20912761362013 @default.
W2091276136 countsByYear W20912761362016 @default.
W2091276136 countsByYear W20912761362019 @default.
W2091276136 countsByYear W20912761362020 @default.
W2091276136 countsByYear W20912761362022 @default.
W2091276136 crossrefType "journal-article" @default.
W2091276136 hasAuthorship W2091276136A5014486140 @default.
W2091276136 hasAuthorship W2091276136A5026193000 @default.
W2091276136 hasAuthorship W2091276136A5052416355 @default.
W2091276136 hasBestOaLocation W20912761361 @default.
W2091276136 hasConcept C104317684 @default.
W2091276136 hasConcept C12554922 @default.
W2091276136 hasConcept C14036430 @default.
W2091276136 hasConcept C143065580 @default.
W2091276136 hasConcept C166342909 @default.
W2091276136 hasConcept C181199279 @default.
W2091276136 hasConcept C185592680 @default.
W2091276136 hasConcept C202751555 @default.
W2091276136 hasConcept C207001950 @default.
W2091276136 hasConcept C54355233 @default.
W2091276136 hasConcept C547475151 @default.
W2091276136 hasConcept C55493867 @default.
W2091276136 hasConcept C70721500 @default.
W2091276136 hasConcept C86803240 @default.
W2091276136 hasConcept C87190427 @default.
W2091276136 hasConcept C95444343 @default.
W2091276136 hasConceptScore W2091276136C104317684 @default.
W2091276136 hasConceptScore W2091276136C12554922 @default.
W2091276136 hasConceptScore W2091276136C14036430 @default.
W2091276136 hasConceptScore W2091276136C143065580 @default.
W2091276136 hasConceptScore W2091276136C166342909 @default.
W2091276136 hasConceptScore W2091276136C181199279 @default.
W2091276136 hasConceptScore W2091276136C185592680 @default.
W2091276136 hasConceptScore W2091276136C202751555 @default.
W2091276136 hasConceptScore W2091276136C207001950 @default.
W2091276136 hasConceptScore W2091276136C54355233 @default.
W2091276136 hasConceptScore W2091276136C547475151 @default.
W2091276136 hasConceptScore W2091276136C55493867 @default.
W2091276136 hasConceptScore W2091276136C70721500 @default.
W2091276136 hasConceptScore W2091276136C86803240 @default.
W2091276136 hasConceptScore W2091276136C87190427 @default.
W2091276136 hasConceptScore W2091276136C95444343 @default.
W2091276136 hasIssue "48" @default.
W2091276136 hasLocation W20912761361 @default.
W2091276136 hasOpenAccess W2091276136 @default.