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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 import of pOTC into rat  liver mitochondria. (a) Rat  pOTC synthesized in the untreated rabbit reticulocyte lysate (Untreated, 15 KBq) or  in the hsc70-depleted lysate  without readdition (Depleted, 2.0 KBq) or with readdition (Depleted + Hsc70,  2.0 KBq) of 1.8 μM mouse  hsc70 before translation, was  subjected to import assay, as  described in Materials and  Methods. (b) Rat pOTC synthesized in the mock-depleted  lysate (Mock-Depleted, 23  KBq) or in the dj2-depleted  lysate without readdition  (Depleted, 20 KBq) or with  readdition (Depleted + His-dj2, 16 KBq) of 0.4 μM His-dj2 before translation was subjected to import assay. (c) Rat pOTC synthesized  in the mock-depleted lysate (Mock-Depleted, 23 KBq) or in the dj1-depleted lysate without readdition (Depleted, 14 KBq) or with readdition (Depleted + His-dj1, 16 KBq) of 0.5 μM His-dj1 before translation was subjected to import assay. Portions of the fluorograms are  shown in the upper part of each panel. p, precursor form of OTC; m, mature form of OTC; 30%, 30% of the input pOTC. The results  were quantitated by imaging plate analysis using FUJIX BAS2000 analyzer and are shown in the lower part.
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Figure 2: Effect of depletion and readdition of chaperones on import of pOTC into rat liver mitochondria. (a) Rat pOTC synthesized in the untreated rabbit reticulocyte lysate (Untreated, 15 KBq) or in the hsc70-depleted lysate without readdition (Depleted, 2.0 KBq) or with readdition (Depleted + Hsc70, 2.0 KBq) of 1.8 μM mouse hsc70 before translation, was subjected to import assay, as described in Materials and Methods. (b) Rat pOTC synthesized in the mock-depleted lysate (Mock-Depleted, 23 KBq) or in the dj2-depleted lysate without readdition (Depleted, 20 KBq) or with readdition (Depleted + His-dj2, 16 KBq) of 0.4 μM His-dj2 before translation was subjected to import assay. (c) Rat pOTC synthesized in the mock-depleted lysate (Mock-Depleted, 23 KBq) or in the dj1-depleted lysate without readdition (Depleted, 14 KBq) or with readdition (Depleted + His-dj1, 16 KBq) of 0.5 μM His-dj1 before translation was subjected to import assay. Portions of the fluorograms are shown in the upper part of each panel. p, precursor form of OTC; m, mature form of OTC; 30%, 30% of the input pOTC. The results were quantitated by imaging plate analysis using FUJIX BAS2000 analyzer and are shown in the lower part.

Mentions: To investigate the roles of cytosolic chaperones in mitochondrial protein import, we synthesized rat pOTC in the untreated or the chaperone(s)-depleted reticulocyte lysate and import assays were done. When hsc70 (and concomitantly dj1) was depleted from the lysate, a decrease in pOTC synthesis was observed (Terada et al., 1995, 1996). The reason for the decrease is unknown. Import of pOTC synthesized in the hsc70-depleted lysate was about one-third that of the precursor synthesized in the untreated lysate (Fig. 2 a). When purified mouse hsc70 (130 μg/ml, 1.8 μM) was readded to the hsc70-depleted lysate before translation, the decreased pOTC import was almost completely recovered. Addition of hsc70 after translation and before import assay was not effective (Terada et al., 1995). Immunoblot analysis ruled out dj1 contamination in our hsc70 preparation (data not shown). These findings indicate a role for hsc70, but not dj1, in pOTC import into mitochondria.


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 import of pOTC into rat  liver mitochondria. (a) Rat  pOTC synthesized in the untreated rabbit reticulocyte lysate (Untreated, 15 KBq) or  in the hsc70-depleted lysate  without readdition (Depleted, 2.0 KBq) or with readdition (Depleted + Hsc70,  2.0 KBq) of 1.8 μM mouse  hsc70 before translation, was  subjected to import assay, as  described in Materials and  Methods. (b) Rat pOTC synthesized in the mock-depleted  lysate (Mock-Depleted, 23  KBq) or in the dj2-depleted  lysate without readdition  (Depleted, 20 KBq) or with  readdition (Depleted + His-dj2, 16 KBq) of 0.4 μM His-dj2 before translation was subjected to import assay. (c) Rat pOTC synthesized  in the mock-depleted lysate (Mock-Depleted, 23 KBq) or in the dj1-depleted lysate without readdition (Depleted, 14 KBq) or with readdition (Depleted + His-dj1, 16 KBq) of 0.5 μM His-dj1 before translation was subjected to import assay. Portions of the fluorograms are  shown in the upper part of each panel. p, precursor form of OTC; m, mature form of OTC; 30%, 30% of the input pOTC. The results  were quantitated by imaging plate analysis using FUJIX BAS2000 analyzer and are shown in the lower part.
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Related In: Results  -  Collection

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Figure 2: Effect of depletion and readdition of chaperones on import of pOTC into rat liver mitochondria. (a) Rat pOTC synthesized in the untreated rabbit reticulocyte lysate (Untreated, 15 KBq) or in the hsc70-depleted lysate without readdition (Depleted, 2.0 KBq) or with readdition (Depleted + Hsc70, 2.0 KBq) of 1.8 μM mouse hsc70 before translation, was subjected to import assay, as described in Materials and Methods. (b) Rat pOTC synthesized in the mock-depleted lysate (Mock-Depleted, 23 KBq) or in the dj2-depleted lysate without readdition (Depleted, 20 KBq) or with readdition (Depleted + His-dj2, 16 KBq) of 0.4 μM His-dj2 before translation was subjected to import assay. (c) Rat pOTC synthesized in the mock-depleted lysate (Mock-Depleted, 23 KBq) or in the dj1-depleted lysate without readdition (Depleted, 14 KBq) or with readdition (Depleted + His-dj1, 16 KBq) of 0.5 μM His-dj1 before translation was subjected to import assay. Portions of the fluorograms are shown in the upper part of each panel. p, precursor form of OTC; m, mature form of OTC; 30%, 30% of the input pOTC. The results were quantitated by imaging plate analysis using FUJIX BAS2000 analyzer and are shown in the lower part.
Mentions: To investigate the roles of cytosolic chaperones in mitochondrial protein import, we synthesized rat pOTC in the untreated or the chaperone(s)-depleted reticulocyte lysate and import assays were done. When hsc70 (and concomitantly dj1) was depleted from the lysate, a decrease in pOTC synthesis was observed (Terada et al., 1995, 1996). The reason for the decrease is unknown. Import of pOTC synthesized in the hsc70-depleted lysate was about one-third that of the precursor synthesized in the untreated lysate (Fig. 2 a). When purified mouse hsc70 (130 μg/ml, 1.8 μM) was readded to the hsc70-depleted lysate before translation, the decreased pOTC import was almost completely recovered. Addition of hsc70 after translation and before import assay was not effective (Terada et al., 1995). Immunoblot analysis ruled out dj1 contamination in our hsc70 preparation (data not shown). These findings indicate a role for hsc70, but not dj1, in pOTC import into mitochondria.

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