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The human DnaJ homologue dj2 facilitates mitochondrial protein import and luciferase refolding.

Terada K, Kanazawa M, Bukau B, Mori M - J. Cell Biol. (1997)

Bottom Line: We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase.Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase.Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan.

ABSTRACT
DnaJ homologues function in cooperation with hsp70 family members in various cellular processes including intracellular protein trafficking and folding. Three human DnaJ homologues present in the cytosol have been identified: dj1 (hsp40/hdj-1), dj2 (HSDJ/hdj-2), and neuronal tissue-specific hsj1. dj1 is thought to be engaged in folding of nascent polypeptides, whereas functions of the other DnaJ homologues remain to be elucidated. To investigate roles of dj2 and dj1, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that heat shock cognate 70 protein (hsc70) and dj2, but not dj1, are involved in mitochondrial import of preornithine transcarbamylase. Bacterial DnaJ could replace mammalian dj2 in mitochondrial protein import. We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase. Unexpectedly, dj2, but not dj1, together with hsc70 refolded the protein efficiently. We propose that dj2 is the functional partner DnaJ homologue of hsc70 in the mammalian cytosol. Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase. Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.

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Intracellular concentrations of  chaperones and depletion for chaperones  of rabbit reticulocyte lysate. (a) Purified  chaperones (0.5 μg each) were analyzed  by SDS-PAGE. Lane 1, hsc70; lane 2,  His-dj2; lane 3, His-dj1; lane 4, DnaJ. (b)  Immunoblot analysis of hsc70 (top), dj2  (middle), and dj1 (bottom) in rabbit reticulocyte lysate (lane 1, 20 μg protein),  HepG2 cell extract (lane 2, 2 μg protein),  COS-7 cell extract (lane 3, 2 μg protein),  and rat liver cytosol (lane 4, 2 μg protein).  Purified chaperones were used as standards (lanes 5–9): top, mouse hsc70 (8, 16,  31, 63 and 130 ng); middle, baculovirus- expressed human His-dj2 (0.63, 1.3, 2.5, 5.0, and 10 ng); bottom, E. coli-expressed human His-dj1 (0.060, 0.13, 0.25, 0.50, and 1.0 ng).  Note that standard dj2 and dj1 had histidine tags and migrated more slowly than the endogenous chaperones in 8% SDS-PAGE. The  reason for the presence of immunoreactive 49K band in rabbit reticulocyte lysate is unknown (middle, lane 1). Human recombinant  hsc70 (a gift from N. Imamoto and Y. Yoneda) and mouse hsc70 gave signals of similar intensities. (c) Immunodepletion was performed  with antibody-coupled Sepharose resins as described in Materials and Methods. Extent of the depletion for the endogenous chaperones was  assessed by immunoblot analysis of the reticulocyte lysates (0.5 μl each, ∼50 μg protein). Protein molecular mass markers (rainbow-colored  markers; Amersham) are myosin (200K), phosphorylase b (97K), serum albumin (69K), ovalbumin (46K), and carbonic anhydrase (30K).
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Figure 1: Intracellular concentrations of chaperones and depletion for chaperones of rabbit reticulocyte lysate. (a) Purified chaperones (0.5 μg each) were analyzed by SDS-PAGE. Lane 1, hsc70; lane 2, His-dj2; lane 3, His-dj1; lane 4, DnaJ. (b) Immunoblot analysis of hsc70 (top), dj2 (middle), and dj1 (bottom) in rabbit reticulocyte lysate (lane 1, 20 μg protein), HepG2 cell extract (lane 2, 2 μg protein), COS-7 cell extract (lane 3, 2 μg protein), and rat liver cytosol (lane 4, 2 μg protein). Purified chaperones were used as standards (lanes 5–9): top, mouse hsc70 (8, 16, 31, 63 and 130 ng); middle, baculovirus- expressed human His-dj2 (0.63, 1.3, 2.5, 5.0, and 10 ng); bottom, E. coli-expressed human His-dj1 (0.060, 0.13, 0.25, 0.50, and 1.0 ng). Note that standard dj2 and dj1 had histidine tags and migrated more slowly than the endogenous chaperones in 8% SDS-PAGE. The reason for the presence of immunoreactive 49K band in rabbit reticulocyte lysate is unknown (middle, lane 1). Human recombinant hsc70 (a gift from N. Imamoto and Y. Yoneda) and mouse hsc70 gave signals of similar intensities. (c) Immunodepletion was performed with antibody-coupled Sepharose resins as described in Materials and Methods. Extent of the depletion for the endogenous chaperones was assessed by immunoblot analysis of the reticulocyte lysates (0.5 μl each, ∼50 μg protein). Protein molecular mass markers (rainbow-colored markers; Amersham) are myosin (200K), phosphorylase b (97K), serum albumin (69K), ovalbumin (46K), and carbonic anhydrase (30K).

Mentions: We purified chaperones including mouse hsc70, human recombinant histidine-tagged dj2 (His-dj2) and dj1 (His-dj1), and Escherichia coli DnaJ (Fig. 1 a). hsc70 migrated as a protein of 73K in SDS–PAGE (lane 1). His-dj2 expressed in Sf9 insect cells gave 49K and 47K polypeptides (lane 2). We have shown that human dj2 is subject to farnesylation at Cys-394 and that the farnesylated form migrates faster than the unfarnesylated form on SDS–polyacrylamide gel (Kanazawa et al., 1997). These two polypeptides also proved to be dj2 by immunoblot analysis. The ratio of unfarnesylated form to farnesylated one was ∼1:2 (Fig. 1 b, middle, lanes 5–9), but this ratio varied from one preparation to another. When His-dj2 expressing insect cells were cultured in the absence of a mevalonate precursor, farnesylation was much decreased (data not shown). On the other hand, human dj1 contains no prenylation motif, and a single 42K polypeptide was observed (Fig. 1 a, lane 3). E. coli DnaJ migrated as a 41K polypeptide (lane 4).


The human DnaJ homologue dj2 facilitates mitochondrial protein import and luciferase refolding.

Terada K, Kanazawa M, Bukau B, Mori M - J. Cell Biol. (1997)

Intracellular concentrations of  chaperones and depletion for chaperones  of rabbit reticulocyte lysate. (a) Purified  chaperones (0.5 μg each) were analyzed  by SDS-PAGE. Lane 1, hsc70; lane 2,  His-dj2; lane 3, His-dj1; lane 4, DnaJ. (b)  Immunoblot analysis of hsc70 (top), dj2  (middle), and dj1 (bottom) in rabbit reticulocyte lysate (lane 1, 20 μg protein),  HepG2 cell extract (lane 2, 2 μg protein),  COS-7 cell extract (lane 3, 2 μg protein),  and rat liver cytosol (lane 4, 2 μg protein).  Purified chaperones were used as standards (lanes 5–9): top, mouse hsc70 (8, 16,  31, 63 and 130 ng); middle, baculovirus- expressed human His-dj2 (0.63, 1.3, 2.5, 5.0, and 10 ng); bottom, E. coli-expressed human His-dj1 (0.060, 0.13, 0.25, 0.50, and 1.0 ng).  Note that standard dj2 and dj1 had histidine tags and migrated more slowly than the endogenous chaperones in 8% SDS-PAGE. The  reason for the presence of immunoreactive 49K band in rabbit reticulocyte lysate is unknown (middle, lane 1). Human recombinant  hsc70 (a gift from N. Imamoto and Y. Yoneda) and mouse hsc70 gave signals of similar intensities. (c) Immunodepletion was performed  with antibody-coupled Sepharose resins as described in Materials and Methods. Extent of the depletion for the endogenous chaperones was  assessed by immunoblot analysis of the reticulocyte lysates (0.5 μl each, ∼50 μg protein). Protein molecular mass markers (rainbow-colored  markers; Amersham) are myosin (200K), phosphorylase b (97K), serum albumin (69K), ovalbumin (46K), and carbonic anhydrase (30K).
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Figure 1: Intracellular concentrations of chaperones and depletion for chaperones of rabbit reticulocyte lysate. (a) Purified chaperones (0.5 μg each) were analyzed by SDS-PAGE. Lane 1, hsc70; lane 2, His-dj2; lane 3, His-dj1; lane 4, DnaJ. (b) Immunoblot analysis of hsc70 (top), dj2 (middle), and dj1 (bottom) in rabbit reticulocyte lysate (lane 1, 20 μg protein), HepG2 cell extract (lane 2, 2 μg protein), COS-7 cell extract (lane 3, 2 μg protein), and rat liver cytosol (lane 4, 2 μg protein). Purified chaperones were used as standards (lanes 5–9): top, mouse hsc70 (8, 16, 31, 63 and 130 ng); middle, baculovirus- expressed human His-dj2 (0.63, 1.3, 2.5, 5.0, and 10 ng); bottom, E. coli-expressed human His-dj1 (0.060, 0.13, 0.25, 0.50, and 1.0 ng). Note that standard dj2 and dj1 had histidine tags and migrated more slowly than the endogenous chaperones in 8% SDS-PAGE. The reason for the presence of immunoreactive 49K band in rabbit reticulocyte lysate is unknown (middle, lane 1). Human recombinant hsc70 (a gift from N. Imamoto and Y. Yoneda) and mouse hsc70 gave signals of similar intensities. (c) Immunodepletion was performed with antibody-coupled Sepharose resins as described in Materials and Methods. Extent of the depletion for the endogenous chaperones was assessed by immunoblot analysis of the reticulocyte lysates (0.5 μl each, ∼50 μg protein). Protein molecular mass markers (rainbow-colored markers; Amersham) are myosin (200K), phosphorylase b (97K), serum albumin (69K), ovalbumin (46K), and carbonic anhydrase (30K).
Mentions: We purified chaperones including mouse hsc70, human recombinant histidine-tagged dj2 (His-dj2) and dj1 (His-dj1), and Escherichia coli DnaJ (Fig. 1 a). hsc70 migrated as a protein of 73K in SDS–PAGE (lane 1). His-dj2 expressed in Sf9 insect cells gave 49K and 47K polypeptides (lane 2). We have shown that human dj2 is subject to farnesylation at Cys-394 and that the farnesylated form migrates faster than the unfarnesylated form on SDS–polyacrylamide gel (Kanazawa et al., 1997). These two polypeptides also proved to be dj2 by immunoblot analysis. The ratio of unfarnesylated form to farnesylated one was ∼1:2 (Fig. 1 b, middle, lanes 5–9), but this ratio varied from one preparation to another. When His-dj2 expressing insect cells were cultured in the absence of a mevalonate precursor, farnesylation was much decreased (data not shown). On the other hand, human dj1 contains no prenylation motif, and a single 42K polypeptide was observed (Fig. 1 a, lane 3). E. coli DnaJ migrated as a 41K polypeptide (lane 4).

Bottom Line: We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase.Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase.Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan.

ABSTRACT
DnaJ homologues function in cooperation with hsp70 family members in various cellular processes including intracellular protein trafficking and folding. Three human DnaJ homologues present in the cytosol have been identified: dj1 (hsp40/hdj-1), dj2 (HSDJ/hdj-2), and neuronal tissue-specific hsj1. dj1 is thought to be engaged in folding of nascent polypeptides, whereas functions of the other DnaJ homologues remain to be elucidated. To investigate roles of dj2 and dj1, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that heat shock cognate 70 protein (hsc70) and dj2, but not dj1, are involved in mitochondrial import of preornithine transcarbamylase. Bacterial DnaJ could replace mammalian dj2 in mitochondrial protein import. We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase. Unexpectedly, dj2, but not dj1, together with hsc70 refolded the protein efficiently. We propose that dj2 is the functional partner DnaJ homologue of hsc70 in the mammalian cytosol. Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase. Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.

Show MeSH
Related in: MedlinePlus