The human DnaJ homologue dj2 facilitates mitochondrial protein import and luciferase refolding - PubMed (original) (raw)
The human DnaJ homologue dj2 facilitates mitochondrial protein import and luciferase refolding
K Terada et al. J Cell Biol. 1997.
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.
Figures
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).
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).
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).
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.
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.
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.
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).
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).
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).
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).
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).
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).
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).
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