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• W2058872839 abstract "DnaJ is an essential cochaperone of mammalian heat shock cognate 70 (hsc70) protein. We previously found that dj2 (HSDJ/hdj-2/rdj1), rather than dj1 (hsp40/hdj-1), is a partner DnaJ for the hsc70-based chaperone system. Here, we compared the distribution of dj1, dj2, and the newly found dj3 (cpr3/DNJ3/HIRIP4/rdj2) in cultured cells. Both dj3 as well as dj2 were farnesylated and were ubiquitously expressed. In immunocytochemical and subfractionation studies, these two proteins colocalized with hsc70 under normal conditions. However, dj1 and hsc70 apparently colocalized in the nucleoli after heat shock. Simultaneous depletion of dj2 and dj3 from rabbit reticulocyte lysate markedly reduced mitochondrial import of pre-ornithine transcarbamylase and refolding of guanidine-denatured luciferase. Re-addition of either dj2 or dj3 led to recovery of these reactions. In a reconstituted system, both hsc70-dj2 and hsc70-dj3 were effective in protein refolding. Anti-apoptotic protein bag-1 further stimulated ATP hydrolysis and protein refolding by both pairs. Thus, dj2 and dj3 are the partner DnaJs of hsc70 within the cell, functionally similar and much more efficient than dj1, and bag-1 is a positive cochaperone of the hsc70-dj2 and hsc70-dj3 systems. DnaJ is an essential cochaperone of mammalian heat shock cognate 70 (hsc70) protein. We previously found that dj2 (HSDJ/hdj-2/rdj1), rather than dj1 (hsp40/hdj-1), is a partner DnaJ for the hsc70-based chaperone system. Here, we compared the distribution of dj1, dj2, and the newly found dj3 (cpr3/DNJ3/HIRIP4/rdj2) in cultured cells. Both dj3 as well as dj2 were farnesylated and were ubiquitously expressed. In immunocytochemical and subfractionation studies, these two proteins colocalized with hsc70 under normal conditions. However, dj1 and hsc70 apparently colocalized in the nucleoli after heat shock. Simultaneous depletion of dj2 and dj3 from rabbit reticulocyte lysate markedly reduced mitochondrial import of pre-ornithine transcarbamylase and refolding of guanidine-denatured luciferase. Re-addition of either dj2 or dj3 led to recovery of these reactions. In a reconstituted system, both hsc70-dj2 and hsc70-dj3 were effective in protein refolding. Anti-apoptotic protein bag-1 further stimulated ATP hydrolysis and protein refolding by both pairs. Thus, dj2 and dj3 are the partner DnaJs of hsc70 within the cell, functionally similar and much more efficient than dj1, and bag-1 is a positive cochaperone of the hsc70-dj2 and hsc70-dj3 systems. heat-shock cognate 70 protein human bag-1M/RAP46 mammalian counterpart of hsp40/hdj-1 mammalian counterpart of HSDJ/hdj-2 mammalian counterpart of CPR3/DNJ3/HIPIR4 H6dj1, H6dj2 and H6dj3, hexahistidine-tagged bag-1 or dj1, dj2, or dj3 pre-ornithine transcarbamylase polymerase chain reaction glutathioneS-transferase kilobase(s) The heat-shock cognate protein 70 (hsc70)1 is a major molecular chaperone present in the mammalian cytosol and mediates various cellular processes, including protein folding and traffic. Among numerous cochaperones for hsc70 (1Johnson J.L. Craig E.A. Cell. 1997; 90: 201-204Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), an essential group is the DnaJ family (2Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (398) Google Scholar). The mammalian dj2 (HSDJ/hdj2/rdj1/hsj2) (3Chellaiah A. Davis A. Mohanakumar T. Biochim. Biophys. Acta. 1993; 1174: 111-113Crossref PubMed Scopus (62) Google Scholar, 4Oh S. Iwahori A. Kato S. Biochim. Biophys. Acta. 1993; 1174: 114-116Crossref PubMed Scopus (59) Google Scholar, 5Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar, 6Royaux I. Minner F. Goffinet A.M. de Rouvroit C.L. Genomics. 1998; 53: 415Crossref PubMed Scopus (4) Google Scholar) and dj3 (cpr3/DNJ3/HIRIP4/rdj2) (5Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar, 7Edwards M.C. Liegeois N. Horecka J. DePinho R.A. Sprague Jr., G.F. Tyers M. Elledge S.J. Genetics. 1997; 147: 1063-1076Crossref PubMed Google Scholar) contain all the domains found in bacterial DnaJ. These orthodox members of the DnaJ subfamily have J-, G/F-, and zinc-finger domains. The zinc-finger domain of bacterial DnaJ coordinates two zinc atoms and is important for binding to denatured protein substrate (8Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (274) Google Scholar). dj2 and dj3 also have the “CAAX” prenylation motif at their COOH termini, and we showed that dj2 is farnesylated (9Kanazawa M. Terada K. Kato S. Mori M. J. Biochem. (Tokyo). 1997; 121: 890-895Crossref PubMed Scopus (64) Google Scholar). The protein sequence of dj3 is 55% identical to the dj2 sequence. dj3, dj2, and hsc70 are all highly conserved among mammals (99% identity between human and rat proteins).On the other hand, dj1 (hsp40/hdj-1) (10Ohtsuka K. Biochem. Biophys. Res. Commun. 1993; 197: 235-240Crossref PubMed Scopus (92) Google Scholar) is a noncanonical member of DnaJ, which lacks the zinc-finger domain. Because dj1 was identified earlier among mammalian DnaJs, many studies of the hsc70 chaperone system were done on dj1. One proposed function for the hsc70-dj1 system is its involvement in folding of nascent polypeptides (11Frydman J. Nimmesgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (562) Google Scholar). Thein vitro protein refolding mediated by the hsc70-dj1 system was also reported (12Freeman B.C. Myers M.P. Schumacher R. Morimoto R.I. EMBO J. 1995; 14: 2281-2292Crossref PubMed Scopus (381) Google Scholar).We previously developed a system of depletion of hsc70, dj1, and dj2 from reticulocyte lysates and of their re-addition, to assess the roles of these chaperones (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar). We found that the hsc70-dj2 chaperone pair is required for mitochondrial import of pre-ornithine transcarbamylase (pOTC) and refolding of chemically denatured luciferase. Unexpectedly, dj1 depletion had little effect on luciferase refolding. A reconstituted system using purified hsc70 and dj2 at physiological concentrations, but not the hsc70-dj1 pair, facilitated luciferase refolding.In the present study, we investigated properties and roles of the newly found dj3 in comparison with those of dj1 and dj2. dj3 as well as dj2 was farnesylated. The pattern of dj1, dj2, and dj3 expression in various tissues was tested at the protein and mRNA levels, as well as the inducibility upon heat shock in cultured cells. Immunofluorescence and organelle fractionation were used to examine the intracellular localization of these DnaJs before and after heat-shock. All these data indicate that dj2 and dj3 are functional partners of hsc70. In chaperone depletion experiments from reticulocyte lysates, dj2 and dj3 were equally effective in facilitating protein import into mitochondria and in luciferase refolding. In reconstitution experiments with purified chaperones, hsc70-dj2 and hsc70-dj3 pairs, but not the hsc70-dj1 pair, were effective in protein refolding. Refolding was further accelerated by bag-1, an anti-apoptotic and grpE-like protein (14Hohfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (336) Google Scholar, 15Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (153) Google Scholar, 16Luders J. Demand J. Schonfelder S. Frien M. Zimmermann R. Hohfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar). Effects of dj1–3 and bag-1 proteins on ATPase activity of hsc70 were also tested. The status of the hsc70-based chaperone cycle on combinations of these cochaperones was discussed. Taken together, hsc70-dj2 and hsc70-dj3 pairs possess much stronger chaperone activities than do the hsc70-dj1 pair in mitochondrial protein import and protein folding under normal conditions.DISCUSSIONIn the present study, we analyzed the roles of recently found dj3 in mitochondrial protein import and protein folding, in comparison with dj1 and dj2. Unexpectedly, we found that our depletion system for dj2 (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar) also depleted dj3 simultaneously. Re-addition experiments revealed that dj3 could replace dj2 in facilitating mitochondrial pOTC import and luciferase refolding. Single depletion for dj3 only resulted in a moderate inhibition, in both assays. dj2 and dj3 enhanced ATPase activity of hsc70 protein. Intracellular localization of these two DnaJs was similar, under normal and heat-shocked conditions. These findings also suggest a functional overlap between dj2 and dj3 in living cells. However, tissue distribution of dj3 mRNA differs from that of dj2 mRNA, although these mRNAs are expressed ubiquitously. Furthermore, dj2 mRNA but not dj3 mRNA is heat-induced. Thus, dj2 and dj3 apparently have similar functions, but their contribution may differ among tissues and depend on existing conditions.Immunocytochemical analysis revealed that dj2 and dj3 proteins colocalized well with hsc70, whereas dj1 did not (Fig. 4 A). Intracellular concentrations of hsc70 protein are several times higher than those of three DnaJs. Thus it is possible that a small portion of hsc70 associates with dj1 in the nucleus, but does not give a significant staining. Interestingly, the staining pattern of bag-1 protein (31Naishiro Y. Adachi M. Okuda H. Yawata A. Mitaka T. Takayama S. Reed J.C. Hinoda Y. Imai K. Oncogene. 1999; 18: 3244-3251Crossref PubMed Scopus (48) Google Scholar) was quite similar to that of hsc70. Heat shock treatment induced marked changes in localization. hsc70 localized in the nucleoli where strong dj1 staining was observed. While dj2 and dj3 localized in a limited perinuclear region, localization of hsc70 was not limited to this region. These results may reflect distinct roles of these DnaJs on hsc70, under different conditions. Under normal conditions, dj2 and dj3 mainly modulate chaperoning activities of hsc70 in the cytoplasm. On the other hand, when hsc70 accumulates in the nucleoli under heat-shocked conditions, hsc70 may be regulated mainly by dj1. Strong heat induction of dj1 mRNA may support this view.The importance of ubiquitously expressed bag-1 on mitochondrial protein import remains to be elucidated. Anti-mouse bag-1 antibody could not detect bag-1 in rabbit reticulocyte lysates, and thus could not assess the roles of bag-1. Interestingly, multiple bag-1-related proteins are present in eukaryotes (32Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). bag-1 accelerated refolding of denatured luciferase together with either hsc70-dj2 or hsc70-dj3 pair (Fig. 7). In contrast, the hsc70-dj1 pair could not support luciferase refolding even in the presence of bag-1. Thus it is clear that hsc70-dj2 and hsc70-dj3 pairs possess stronger chaperone activities than do the hsc70-dj1 pair. The zinc-finger domain of orthodox DnaJ proteins may be necessary for the strength of these activities. dj1 was initially reported to maintain folding competence of partially denatured luciferase by heat, and one or more factors in the reticulocyte lysate were required for subsequent folding (30Minami Y. Hohfeld J. Ohtsuka K. Hartl F.U. J. Biol. Chem. 1996; 271: 19617-19624Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Hip and bag-1 have been postulated to be candidates for regulating hsc70-dj1 pair (16Luders J. Demand J. Schonfelder S. Frien M. Zimmermann R. Hohfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar, 32Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). A high level of bag-1 protein apparently inhibited refolding of thermally denatured luciferase in the presence of hsc70-dj1. Contrary to these published data, we found that 0.4 μm bag-1 accelerates chaperone activity of hsc70-dj2 and hsc70-dj3 systems. The physiological concentration of bag-1 in various cell lines was determined to be 0–0.04% of total protein (33Takayama S. Krajewski S. Krajewska M. Kitada S. Zapata J.M. Kochel K. Knee D. Scudiero D. Tudor G. Miller G.J. Miyashita T. Yamada M. Reed J.C. Cancer Res. 1998; 58: 3116-3131PubMed Google Scholar). Negative effects of bag-1 could be caused by excessive amounts over hsc70 in vitro. It would be of interest to test other cochaperones on our hsc70-dj2 and hsc70-dj3 pairs.Refolding of luciferase in the reconstituted system corresponded well with ATP hydrolysis (Figs. 7 and 8). Our data suggest that the status of hsc70-based chaperone cycle depends on combinations of cochaperones. When hsc70 alone was used, no chaperone activity was observed. Combination of hsc70 and bag-1 had no effect. In the presence of dj2 or dj3, ATP hydrolysis was accelerated, and hsc70-dj2 and hsc70-dj3 pairs began to exhibit their chaperone function. However, ADP release from hsc70 is still the rate-limiting step for these pairs. Further addition of bag-1 accelerated ADP release and fully activated hsc70-based chaperone cycle.We found that dj2 is the most efficient partner of hsc70 with regard to mitochondrial protein import and protein refolding (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar). Recently, dj2 was shown to facilitate early steps of assembly of the cystic fibrosis transmembrane conductance regulator in the endoplasmic reticulum (24Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (267) Google Scholar). Involvement of the hsc70-dj2 chaperone pair in polyglutamine repeat disorders (34Cummings C.J. Mancini M.A. Antalffy B. DeFranco D.B. Orr H.T. Zoghbi H.Y. Nat. Genet. 1998; 19: 148-154Crossref PubMed Scopus (747) Google Scholar, 35Stenoien D.L. Cummings C.J. Adams H.P. Mancini M.G. Patel K. DeMartino G.N. Marcelli M. Weigel N.L. Mancini M.A. Hum. Mol. Genet. 1999; 8: 731-741Crossref PubMed Scopus (389) Google Scholar) and in defects of a mutant glucocorticoid receptor (36Tang Y. Ramakrishnan C. Thomas J. DeFranco D.B. Mol. Biol. Cell. 1997; 8: 795-809Crossref PubMed Scopus (44) Google Scholar) has also been documented. It remains to be tested whether dj3 can replace proposed functions of dj2. On the other hand, dj3 was found to be a farnesylated protein in a rat cDNA library (5Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar) and in a human gene, which restored cell cycle progression in yeast (7Edwards M.C. Liegeois N. Horecka J. DePinho R.A. Sprague Jr., G.F. Tyers M. Elledge S.J. Genetics. 1997; 147: 1063-1076Crossref PubMed Google Scholar). It also remains to be tested whether dj2 can suppress cell cycle defect in yeast mutant. We are now disrupting genes for these DnaJs to better understand the precise role(s) of respective DnaJs in vivo. The heat-shock cognate protein 70 (hsc70)1 is a major molecular chaperone present in the mammalian cytosol and mediates various cellular processes, including protein folding and traffic. Among numerous cochaperones for hsc70 (1Johnson J.L. Craig E.A. Cell. 1997; 90: 201-204Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), an essential group is the DnaJ family (2Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (398) Google Scholar). The mammalian dj2 (HSDJ/hdj2/rdj1/hsj2) (3Chellaiah A. Davis A. Mohanakumar T. Biochim. Biophys. Acta. 1993; 1174: 111-113Crossref PubMed Scopus (62) Google Scholar, 4Oh S. Iwahori A. Kato S. Biochim. Biophys. Acta. 1993; 1174: 114-116Crossref PubMed Scopus (59) Google Scholar, 5Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar, 6Royaux I. Minner F. Goffinet A.M. de Rouvroit C.L. Genomics. 1998; 53: 415Crossref PubMed Scopus (4) Google Scholar) and dj3 (cpr3/DNJ3/HIRIP4/rdj2) (5Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar, 7Edwards M.C. Liegeois N. Horecka J. DePinho R.A. Sprague Jr., G.F. Tyers M. Elledge S.J. Genetics. 1997; 147: 1063-1076Crossref PubMed Google Scholar) contain all the domains found in bacterial DnaJ. These orthodox members of the DnaJ subfamily have J-, G/F-, and zinc-finger domains. The zinc-finger domain of bacterial DnaJ coordinates two zinc atoms and is important for binding to denatured protein substrate (8Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (274) Google Scholar). dj2 and dj3 also have the “CAAX” prenylation motif at their COOH termini, and we showed that dj2 is farnesylated (9Kanazawa M. Terada K. Kato S. Mori M. J. Biochem. (Tokyo). 1997; 121: 890-895Crossref PubMed Scopus (64) Google Scholar). The protein sequence of dj3 is 55% identical to the dj2 sequence. dj3, dj2, and hsc70 are all highly conserved among mammals (99% identity between human and rat proteins). On the other hand, dj1 (hsp40/hdj-1) (10Ohtsuka K. Biochem. Biophys. Res. Commun. 1993; 197: 235-240Crossref PubMed Scopus (92) Google Scholar) is a noncanonical member of DnaJ, which lacks the zinc-finger domain. Because dj1 was identified earlier among mammalian DnaJs, many studies of the hsc70 chaperone system were done on dj1. One proposed function for the hsc70-dj1 system is its involvement in folding of nascent polypeptides (11Frydman J. Nimmesgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (562) Google Scholar). Thein vitro protein refolding mediated by the hsc70-dj1 system was also reported (12Freeman B.C. Myers M.P. Schumacher R. Morimoto R.I. EMBO J. 1995; 14: 2281-2292Crossref PubMed Scopus (381) Google Scholar). We previously developed a system of depletion of hsc70, dj1, and dj2 from reticulocyte lysates and of their re-addition, to assess the roles of these chaperones (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar). We found that the hsc70-dj2 chaperone pair is required for mitochondrial import of pre-ornithine transcarbamylase (pOTC) and refolding of chemically denatured luciferase. Unexpectedly, dj1 depletion had little effect on luciferase refolding. A reconstituted system using purified hsc70 and dj2 at physiological concentrations, but not the hsc70-dj1 pair, facilitated luciferase refolding. In the present study, we investigated properties and roles of the newly found dj3 in comparison with those of dj1 and dj2. dj3 as well as dj2 was farnesylated. The pattern of dj1, dj2, and dj3 expression in various tissues was tested at the protein and mRNA levels, as well as the inducibility upon heat shock in cultured cells. Immunofluorescence and organelle fractionation were used to examine the intracellular localization of these DnaJs before and after heat-shock. All these data indicate that dj2 and dj3 are functional partners of hsc70. In chaperone depletion experiments from reticulocyte lysates, dj2 and dj3 were equally effective in facilitating protein import into mitochondria and in luciferase refolding. In reconstitution experiments with purified chaperones, hsc70-dj2 and hsc70-dj3 pairs, but not the hsc70-dj1 pair, were effective in protein refolding. Refolding was further accelerated by bag-1, an anti-apoptotic and grpE-like protein (14Hohfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (336) Google Scholar, 15Bimston D. Song J. Winchester D. Takayama S. Reed J.C. Morimoto R.I. EMBO J. 1998; 17: 6871-6878Crossref PubMed Scopus (153) Google Scholar, 16Luders J. Demand J. Schonfelder S. Frien M. Zimmermann R. Hohfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar). Effects of dj1–3 and bag-1 proteins on ATPase activity of hsc70 were also tested. The status of the hsc70-based chaperone cycle on combinations of these cochaperones was discussed. Taken together, hsc70-dj2 and hsc70-dj3 pairs possess much stronger chaperone activities than do the hsc70-dj1 pair in mitochondrial protein import and protein folding under normal conditions. DISCUSSIONIn the present study, we analyzed the roles of recently found dj3 in mitochondrial protein import and protein folding, in comparison with dj1 and dj2. Unexpectedly, we found that our depletion system for dj2 (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar) also depleted dj3 simultaneously. Re-addition experiments revealed that dj3 could replace dj2 in facilitating mitochondrial pOTC import and luciferase refolding. Single depletion for dj3 only resulted in a moderate inhibition, in both assays. dj2 and dj3 enhanced ATPase activity of hsc70 protein. Intracellular localization of these two DnaJs was similar, under normal and heat-shocked conditions. These findings also suggest a functional overlap between dj2 and dj3 in living cells. However, tissue distribution of dj3 mRNA differs from that of dj2 mRNA, although these mRNAs are expressed ubiquitously. Furthermore, dj2 mRNA but not dj3 mRNA is heat-induced. Thus, dj2 and dj3 apparently have similar functions, but their contribution may differ among tissues and depend on existing conditions.Immunocytochemical analysis revealed that dj2 and dj3 proteins colocalized well with hsc70, whereas dj1 did not (Fig. 4 A). Intracellular concentrations of hsc70 protein are several times higher than those of three DnaJs. Thus it is possible that a small portion of hsc70 associates with dj1 in the nucleus, but does not give a significant staining. Interestingly, the staining pattern of bag-1 protein (31Naishiro Y. Adachi M. Okuda H. Yawata A. Mitaka T. Takayama S. Reed J.C. Hinoda Y. Imai K. Oncogene. 1999; 18: 3244-3251Crossref PubMed Scopus (48) Google Scholar) was quite similar to that of hsc70. Heat shock treatment induced marked changes in localization. hsc70 localized in the nucleoli where strong dj1 staining was observed. While dj2 and dj3 localized in a limited perinuclear region, localization of hsc70 was not limited to this region. These results may reflect distinct roles of these DnaJs on hsc70, under different conditions. Under normal conditions, dj2 and dj3 mainly modulate chaperoning activities of hsc70 in the cytoplasm. On the other hand, when hsc70 accumulates in the nucleoli under heat-shocked conditions, hsc70 may be regulated mainly by dj1. Strong heat induction of dj1 mRNA may support this view.The importance of ubiquitously expressed bag-1 on mitochondrial protein import remains to be elucidated. Anti-mouse bag-1 antibody could not detect bag-1 in rabbit reticulocyte lysates, and thus could not assess the roles of bag-1. Interestingly, multiple bag-1-related proteins are present in eukaryotes (32Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). bag-1 accelerated refolding of denatured luciferase together with either hsc70-dj2 or hsc70-dj3 pair (Fig. 7). In contrast, the hsc70-dj1 pair could not support luciferase refolding even in the presence of bag-1. Thus it is clear that hsc70-dj2 and hsc70-dj3 pairs possess stronger chaperone activities than do the hsc70-dj1 pair. The zinc-finger domain of orthodox DnaJ proteins may be necessary for the strength of these activities. dj1 was initially reported to maintain folding competence of partially denatured luciferase by heat, and one or more factors in the reticulocyte lysate were required for subsequent folding (30Minami Y. Hohfeld J. Ohtsuka K. Hartl F.U. J. Biol. Chem. 1996; 271: 19617-19624Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Hip and bag-1 have been postulated to be candidates for regulating hsc70-dj1 pair (16Luders J. Demand J. Schonfelder S. Frien M. Zimmermann R. Hohfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar, 32Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). A high level of bag-1 protein apparently inhibited refolding of thermally denatured luciferase in the presence of hsc70-dj1. Contrary to these published data, we found that 0.4 μm bag-1 accelerates chaperone activity of hsc70-dj2 and hsc70-dj3 systems. The physiological concentration of bag-1 in various cell lines was determined to be 0–0.04% of total protein (33Takayama S. Krajewski S. Krajewska M. Kitada S. Zapata J.M. Kochel K. Knee D. Scudiero D. Tudor G. Miller G.J. Miyashita T. Yamada M. Reed J.C. Cancer Res. 1998; 58: 3116-3131PubMed Google Scholar). Negative effects of bag-1 could be caused by excessive amounts over hsc70 in vitro. It would be of interest to test other cochaperones on our hsc70-dj2 and hsc70-dj3 pairs.Refolding of luciferase in the reconstituted system corresponded well with ATP hydrolysis (Figs. 7 and 8). Our data suggest that the status of hsc70-based chaperone cycle depends on combinations of cochaperones. When hsc70 alone was used, no chaperone activity was observed. Combination of hsc70 and bag-1 had no effect. In the presence of dj2 or dj3, ATP hydrolysis was accelerated, and hsc70-dj2 and hsc70-dj3 pairs began to exhibit their chaperone function. However, ADP release from hsc70 is still the rate-limiting step for these pairs. Further addition of bag-1 accelerated ADP release and fully activated hsc70-based chaperone cycle.We found that dj2 is the most efficient partner of hsc70 with regard to mitochondrial protein import and protein refolding (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar). Recently, dj2 was shown to facilitate early steps of assembly of the cystic fibrosis transmembrane conductance regulator in the endoplasmic reticulum (24Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (267) Google Scholar). Involvement of the hsc70-dj2 chaperone pair in polyglutamine repeat disorders (34Cummings C.J. Mancini M.A. Antalffy B. DeFranco D.B. Orr H.T. Zoghbi H.Y. Nat. Genet. 1998; 19: 148-154Crossref PubMed Scopus (747) Google Scholar, 35Stenoien D.L. Cummings C.J. Adams H.P. Mancini M.G. Patel K. DeMartino G.N. Marcelli M. Weigel N.L. Mancini M.A. Hum. Mol. Genet. 1999; 8: 731-741Crossref PubMed Scopus (389) Google Scholar) and in defects of a mutant glucocorticoid receptor (36Tang Y. Ramakrishnan C. Thomas J. DeFranco D.B. Mol. Biol. Cell. 1997; 8: 795-809Crossref PubMed Scopus (44) Google Scholar) has also been documented. It remains to be tested whether dj3 can replace proposed functions of dj2. On the other hand, dj3 was found to be a farnesylated protein in a rat cDNA library (5Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar) and in a human gene, which restored cell cycle progression in yeast (7Edwards M.C. Liegeois N. Horecka J. DePinho R.A. Sprague Jr., G.F. Tyers M. Elledge S.J. Genetics. 1997; 147: 1063-1076Crossref PubMed Google Scholar). It also remains to be tested whether dj2 can suppress cell cycle defect in yeast mutant. We are now disrupting genes for these DnaJs to better understand the precise role(s) of respective DnaJs in vivo. In the present study, we analyzed the roles of recently found dj3 in mitochondrial protein import and protein folding, in comparison with dj1 and dj2. Unexpectedly, we found that our depletion system for dj2 (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar) also depleted dj3 simultaneously. Re-addition experiments revealed that dj3 could replace dj2 in facilitating mitochondrial pOTC import and luciferase refolding. Single depletion for dj3 only resulted in a moderate inhibition, in both assays. dj2 and dj3 enhanced ATPase activity of hsc70 protein. Intracellular localization of these two DnaJs was similar, under normal and heat-shocked conditions. These findings also suggest a functional overlap between dj2 and dj3 in living cells. However, tissue distribution of dj3 mRNA differs from that of dj2 mRNA, although these mRNAs are expressed ubiquitously. Furthermore, dj2 mRNA but not dj3 mRNA is heat-induced. Thus, dj2 and dj3 apparently have similar functions, but their contribution may differ among tissues and depend on existing conditions. Immunocytochemical analysis revealed that dj2 and dj3 proteins colocalized well with hsc70, whereas dj1 did not (Fig. 4 A). Intracellular concentrations of hsc70 protein are several times higher than those of three DnaJs. Thus it is possible that a small portion of hsc70 associates with dj1 in the nucleus, but does not give a significant staining. Interestingly, the staining pattern of bag-1 protein (31Naishiro Y. Adachi M. Okuda H. Yawata A. Mitaka T. Takayama S. Reed J.C. Hinoda Y. Imai K. Oncogene. 1999; 18: 3244-3251Crossref PubMed Scopus (48) Google Scholar) was quite similar to that of hsc70. Heat shock treatment induced marked changes in localization. hsc70 localized in the nucleoli where strong dj1 staining was observed. While dj2 and dj3 localized in a limited perinuclear region, localization of hsc70 was not limited to this region. These results may reflect distinct roles of these DnaJs on hsc70, under different conditions. Under normal conditions, dj2 and dj3 mainly modulate chaperoning activities of hsc70 in the cytoplasm. On the other hand, when hsc70 accumulates in the nucleoli under heat-shocked conditions, hsc70 may be regulated mainly by dj1. Strong heat induction of dj1 mRNA may support this view. The importance of ubiquitously expressed bag-1 on mitochondrial protein import remains to be elucidated. Anti-mouse bag-1 antibody could not detect bag-1 in rabbit reticulocyte lysates, and thus could not assess the roles of bag-1. Interestingly, multiple bag-1-related proteins are present in eukaryotes (32Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). bag-1 accelerated refolding of denatured luciferase together with either hsc70-dj2 or hsc70-dj3 pair (Fig. 7). In contrast, the hsc70-dj1 pair could not support luciferase refolding even in the presence of bag-1. Thus it is clear that hsc70-dj2 and hsc70-dj3 pairs possess stronger chaperone activities than do the hsc70-dj1 pair. The zinc-finger domain of orthodox DnaJ proteins may be necessary for the strength of these activities. dj1 was initially reported to maintain folding competence of partially denatured luciferase by heat, and one or more factors in the reticulocyte lysate were required for subsequent folding (30Minami Y. Hohfeld J. Ohtsuka K. Hartl F.U. J. Biol. Chem. 1996; 271: 19617-19624Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Hip and bag-1 have been postulated to be candidates for regulating hsc70-dj1 pair (16Luders J. Demand J. Schonfelder S. Frien M. Zimmermann R. Hohfeld J. Biol. Chem. 1998; 379: 1217-1226Crossref PubMed Scopus (34) Google Scholar, 32Takayama S. Xie Z. Reed J.C. J. Biol. Chem. 1999; 274: 781-786Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). A high level of bag-1 protein apparently inhibited refolding of thermally denatured luciferase in the presence of hsc70-dj1. Contrary to these published data, we found that 0.4 μm bag-1 accelerates chaperone activity of hsc70-dj2 and hsc70-dj3 systems. The physiological concentration of bag-1 in various cell lines was determined to be 0–0.04% of total protein (33Takayama S. Krajewski S. Krajewska M. Kitada S. Zapata J.M. Kochel K. Knee D. Scudiero D. Tudor G. Miller G.J. Miyashita T. Yamada M. Reed J.C. Cancer Res. 1998; 58: 3116-3131PubMed Google Scholar). Negative effects of bag-1 could be caused by excessive amounts over hsc70 in vitro. It would be of interest to test other cochaperones on our hsc70-dj2 and hsc70-dj3 pairs. Refolding of luciferase in the reconstituted system corresponded well with ATP hydrolysis (Figs. 7 and 8). Our data suggest that the status of hsc70-based chaperone cycle depends on combinations of cochaperones. When hsc70 alone was used, no chaperone activity was observed. Combination of hsc70 and bag-1 had no effect. In the presence of dj2 or dj3, ATP hydrolysis was accelerated, and hsc70-dj2 and hsc70-dj3 pairs began to exhibit their chaperone function. However, ADP release from hsc70 is still the rate-limiting step for these pairs. Further addition of bag-1 accelerated ADP release and fully activated hsc70-based chaperone cycle. We found that dj2 is the most efficient partner of hsc70 with regard to mitochondrial protein import and protein refolding (13Terada K. Kanazawa M. Bukau B. Mori M. J. Cell Biol. 1997; 139: 1089-1095Crossref PubMed Scopus (96) Google Scholar). Recently, dj2 was shown to facilitate early steps of assembly of the cystic fibrosis transmembrane conductance regulator in the endoplasmic reticulum (24Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (267) Google Scholar). Involvement of the hsc70-dj2 chaperone pair in polyglutamine repeat disorders (34Cummings C.J. Mancini M.A. Antalffy B. DeFranco D.B. Orr H.T. Zoghbi H.Y. Nat. Genet. 1998; 19: 148-154Crossref PubMed Scopus (747) Google Scholar, 35Stenoien D.L. Cummings C.J. Adams H.P. Mancini M.G. Patel K. DeMartino G.N. Marcelli M. Weigel N.L. Mancini M.A. Hum. Mol. Genet. 1999; 8: 731-741Crossref PubMed Scopus (389) Google Scholar) and in defects of a mutant glucocorticoid receptor (36Tang Y. Ramakrishnan C. Thomas J. DeFranco D.B. Mol. Biol. Cell. 1997; 8: 795-809Crossref PubMed Scopus (44) Google Scholar) has also been documented. It remains to be tested whether dj3 can replace proposed functions of dj2. On the other hand, dj3 was found to be a farnesylated protein in a rat cDNA library (5Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar) and in a human gene, which restored cell cycle progression in yeast (7Edwards M.C. Liegeois N. Horecka J. DePinho R.A. Sprague Jr., G.F. Tyers M. Elledge S.J. Genetics. 1997; 147: 1063-1076Crossref PubMed Google Scholar). It also remains to be tested whether dj2 can suppress cell cycle defect in yeast mutant. We are now disrupting genes for these DnaJs to better understand the precise role(s) of respective DnaJs in vivo. We thank M. Takiguchi for the gift of human liver cDNA, colleagues of our laboratory for suggestions and technical advice, and M. Ohara for comments on the manuscript." @default.
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• W2058872839 title "Human DnaJ Homologs dj2 and dj3, and bag-1 Are Positive Cochaperones of hsc70" @default.
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