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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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Effect of depletion  and readdition of chaperones  on refolding of chemically denatured luciferase. (a) Chemically denatured luciferase was  renatured in the untreated rabbit reticulocyte lysate (Untreated) or in the hsc70-depleted  lysate without readdition (Depleted) or with readdition (Depleted + Hsc70) of 1.8 μM  mouse hsc70. For comparison,  luciferase was renatured in the  absence of the lysate (Buffer).  (b) Denatured luciferase was  renatured in the mock-depleted  lysate (Mock-Depleted) or in  the dj2-depleted lysate without  readdition (Depleted) or with  readdition (Depleted + His-dj2)  of 0.2 μM His-dj2. Luciferase  was also renatured in the absence of the lysate (Buffer). (c)  Denatured luciferase was renatured in the mock-depleted lysate (Mock-Depleted) or in the dj1-depleted lysate without readdition (Depleted) or with readdition (Depleted + His-dj1) of 0.4 μM His-dj1. Luciferase was also renatured in the absence of the lysate (Buffer). (d) Refolding of luciferase was  conducted for 60 min at 25°C in the dj2-depleted lysate supplemented with decreasing amounts of His-dj2 (0.4, 0.2, 0.1, and 0.03 μM) or  E. coli DnaJ (0.4, 0.2, 0.1, and 0.03 μM). (e) Refolding of luciferase was conducted for 90 min in the hsc70-depleted lysate supplemented  with mouse hsc70 (1.8 μM), His-dj2 (0.4 μM), His-dj1 (0.5 μM), or DnaJ (0.5 μM).
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Figure 3: Effect of depletion and readdition of chaperones on refolding of chemically denatured luciferase. (a) Chemically denatured luciferase was renatured in the untreated rabbit reticulocyte lysate (Untreated) or in the hsc70-depleted lysate without readdition (Depleted) or with readdition (Depleted + Hsc70) of 1.8 μM mouse hsc70. For comparison, luciferase was renatured in the absence of the lysate (Buffer). (b) Denatured luciferase was renatured in the mock-depleted lysate (Mock-Depleted) or in the dj2-depleted lysate without readdition (Depleted) or with readdition (Depleted + His-dj2) of 0.2 μM His-dj2. Luciferase was also renatured in the absence of the lysate (Buffer). (c) Denatured luciferase was renatured in the mock-depleted lysate (Mock-Depleted) or in the dj1-depleted lysate without readdition (Depleted) or with readdition (Depleted + His-dj1) of 0.4 μM His-dj1. Luciferase was also renatured in the absence of the lysate (Buffer). (d) Refolding of luciferase was conducted for 60 min at 25°C in the dj2-depleted lysate supplemented with decreasing amounts of His-dj2 (0.4, 0.2, 0.1, and 0.03 μM) or E. coli DnaJ (0.4, 0.2, 0.1, and 0.03 μM). (e) Refolding of luciferase was conducted for 90 min in the hsc70-depleted lysate supplemented with mouse hsc70 (1.8 μM), His-dj2 (0.4 μM), His-dj1 (0.5 μM), or DnaJ (0.5 μM).

Mentions: Chemically denatured luciferase when incubated alone at 25°C was not efficiently refolded into the enzymatically active form (Fig. 3 a). On the other hand, it was efficiently refolded in the untreated reticulocyte lysate. Refolding of luciferase proceeded with time and reached a plateau within 60 min. 87% of the enzyme activity was recovered in 90 min. These results are in good accord with the documented data (Nimmesgern and Hartl, 1993). When the reaction was conducted in the hsc70-depleted lysate, refolding was markedly decreased. The residual low refolding activity in the hsc70-depleted lysate may be attributed to remaining chaperones other than hsc70 and dj1. When purified hsc70 (1.8 μM) was supplemented to the hsc70- depleted lysate before the refolding assay, the refolding activity recovered almost completely. It is to be noted that this recovery was independent of dj1 that was also removed from the lysate by the hsc70-depletion procedure (see above).


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

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

Effect of depletion  and readdition of chaperones  on refolding of chemically denatured luciferase. (a) Chemically denatured luciferase was  renatured in the untreated rabbit reticulocyte lysate (Untreated) or in the hsc70-depleted  lysate without readdition (Depleted) or with readdition (Depleted + Hsc70) of 1.8 μM  mouse hsc70. For comparison,  luciferase was renatured in the  absence of the lysate (Buffer).  (b) Denatured luciferase was  renatured in the mock-depleted  lysate (Mock-Depleted) or in  the dj2-depleted lysate without  readdition (Depleted) or with  readdition (Depleted + His-dj2)  of 0.2 μM His-dj2. Luciferase  was also renatured in the absence of the lysate (Buffer). (c)  Denatured luciferase was renatured in the mock-depleted lysate (Mock-Depleted) or in the dj1-depleted lysate without readdition (Depleted) or with readdition (Depleted + His-dj1) of 0.4 μM His-dj1. Luciferase was also renatured in the absence of the lysate (Buffer). (d) Refolding of luciferase was  conducted for 60 min at 25°C in the dj2-depleted lysate supplemented with decreasing amounts of His-dj2 (0.4, 0.2, 0.1, and 0.03 μM) or  E. coli DnaJ (0.4, 0.2, 0.1, and 0.03 μM). (e) Refolding of luciferase was conducted for 90 min in the hsc70-depleted lysate supplemented  with mouse hsc70 (1.8 μM), His-dj2 (0.4 μM), His-dj1 (0.5 μM), or DnaJ (0.5 μM).
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Figure 3: Effect of depletion and readdition of chaperones on refolding of chemically denatured luciferase. (a) Chemically denatured luciferase was renatured in the untreated rabbit reticulocyte lysate (Untreated) or in the hsc70-depleted lysate without readdition (Depleted) or with readdition (Depleted + Hsc70) of 1.8 μM mouse hsc70. For comparison, luciferase was renatured in the absence of the lysate (Buffer). (b) Denatured luciferase was renatured in the mock-depleted lysate (Mock-Depleted) or in the dj2-depleted lysate without readdition (Depleted) or with readdition (Depleted + His-dj2) of 0.2 μM His-dj2. Luciferase was also renatured in the absence of the lysate (Buffer). (c) Denatured luciferase was renatured in the mock-depleted lysate (Mock-Depleted) or in the dj1-depleted lysate without readdition (Depleted) or with readdition (Depleted + His-dj1) of 0.4 μM His-dj1. Luciferase was also renatured in the absence of the lysate (Buffer). (d) Refolding of luciferase was conducted for 60 min at 25°C in the dj2-depleted lysate supplemented with decreasing amounts of His-dj2 (0.4, 0.2, 0.1, and 0.03 μM) or E. coli DnaJ (0.4, 0.2, 0.1, and 0.03 μM). (e) Refolding of luciferase was conducted for 90 min in the hsc70-depleted lysate supplemented with mouse hsc70 (1.8 μM), His-dj2 (0.4 μM), His-dj1 (0.5 μM), or DnaJ (0.5 μM).
Mentions: Chemically denatured luciferase when incubated alone at 25°C was not efficiently refolded into the enzymatically active form (Fig. 3 a). On the other hand, it was efficiently refolded in the untreated reticulocyte lysate. Refolding of luciferase proceeded with time and reached a plateau within 60 min. 87% of the enzyme activity was recovered in 90 min. These results are in good accord with the documented data (Nimmesgern and Hartl, 1993). When the reaction was conducted in the hsc70-depleted lysate, refolding was markedly decreased. The residual low refolding activity in the hsc70-depleted lysate may be attributed to remaining chaperones other than hsc70 and dj1. When purified hsc70 (1.8 μM) was supplemented to the hsc70- depleted lysate before the refolding assay, the refolding activity recovered almost completely. It is to be noted that this recovery was independent of dj1 that was also removed from the lysate by the hsc70-depletion procedure (see above).

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