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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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Refolding of luciferase by purified chaperones. (a)  Chemically denatured luciferase was renatured for 90 min at 25°C  in the presence of each chaperone or their combinations. Concentrations of proteins added were: hsc70, 1.8 μM; His-dj2, 0.4  μM; His-dj1, 0.5 μM; DnaJ, 0.5 μM; bovine serum albumin, 1.8  μM. (b) Refolding was performed for indicated periods in the  presence of indicated chaperone(s).
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Figure 4: Refolding of luciferase by purified chaperones. (a) Chemically denatured luciferase was renatured for 90 min at 25°C in the presence of each chaperone or their combinations. Concentrations of proteins added were: hsc70, 1.8 μM; His-dj2, 0.4 μM; His-dj1, 0.5 μM; DnaJ, 0.5 μM; bovine serum albumin, 1.8 μM. (b) Refolding was performed for indicated periods in the presence of indicated chaperone(s).

Mentions: Refolding of luciferase by these chaperones was studied in greater detail using purified components. As shown in Fig. 4 a, luciferase refolding was somewhat facilitated by hsc70, His-dj2, and His-dj1. However, bovine serum albumin at a similar concentration was also effective. Therefore, the facilitation effects by these chaperones do not appear to be due to their specific chaperone activities. On the other hand, luciferase activity could be recovered by the addition of either His-dj2 or DnaJ together with hsc70. The addition of His-dj1 together with hsc70 had no significant effect.


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

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

Refolding of luciferase by purified chaperones. (a)  Chemically denatured luciferase was renatured for 90 min at 25°C  in the presence of each chaperone or their combinations. Concentrations of proteins added were: hsc70, 1.8 μM; His-dj2, 0.4  μM; His-dj1, 0.5 μM; DnaJ, 0.5 μM; bovine serum albumin, 1.8  μM. (b) Refolding was performed for indicated periods in the  presence of indicated chaperone(s).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140199&req=5

Figure 4: Refolding of luciferase by purified chaperones. (a) Chemically denatured luciferase was renatured for 90 min at 25°C in the presence of each chaperone or their combinations. Concentrations of proteins added were: hsc70, 1.8 μM; His-dj2, 0.4 μM; His-dj1, 0.5 μM; DnaJ, 0.5 μM; bovine serum albumin, 1.8 μM. (b) Refolding was performed for indicated periods in the presence of indicated chaperone(s).
Mentions: Refolding of luciferase by these chaperones was studied in greater detail using purified components. As shown in Fig. 4 a, luciferase refolding was somewhat facilitated by hsc70, His-dj2, and His-dj1. However, bovine serum albumin at a similar concentration was also effective. Therefore, the facilitation effects by these chaperones do not appear to be due to their specific chaperone activities. On the other hand, luciferase activity could be recovered by the addition of either His-dj2 or DnaJ together with hsc70. The addition of His-dj1 together with hsc70 had no significant effect.

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