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Mitochondrial preproteins are synthesized in the cytosol with N-terminal signal sequences (presequences) or internal targeting signals. Generally, preproteins with presequences are initially recognized by Tom20 (translocase of the outer membrane) and, subsequently, by Tom22, whereas hydrophobic preproteins with internal targeting signals are first recognized by Tom70. Recent studies suggest that Tom70 associates with molecular chaperones, thereby maintaining their substrate preproteins in an import-competent state. However, such a function has not been reported for other Tom component(s). Here, we investigated a role for Tom20 in preventing substrate preproteins from aggregating. In vitro binding assays showed that Tom20 binds to guanidinium chloride unfolded substrate proteins regardless of the presence or absence of presequences. This suggests that Tom20 functions as a receptor not only for presequences but also for mature portions exposed in unfolded preproteins. Aggregation suppression assays on citrate synthase showed that the cytosolic domain of Tom20 has a chaperone-like activity to prevent this protein from aggregating. This activity was inhibited by a presequence peptide, suggesting that the binding site of Tom20 for presequence is identical or close to the active site for the chaperone-like activity. The cytosolic domain of Tom22 also showed a similar activity for citrate synthase, whereas Tom70 did not. These results suggest that the cytosolic domains of Tom20 and Tom22 function to maintain their substrate preproteins unfolded and prevent them from aggregating on the mitochondrial surface.

Carbon tetrachloride (CCl4) induces liver damage, apparently through the formation of free-radical metabolites. Molecular chaperones such as heat shock protein (Hsp) of 70 kDa have been found to protect cells from various stresses. We previously found that cytosolic chaperone pairs of the Hsp70 family and their DnaJ homolog cochaperones prevent nitric oxide-mediated apoptosis and heat-induced cell death. Expression of cytosolic chaperones, including Hsp70; heat shock cognate (Hsc) 70; and DnaJ homologs dj1 (DjB1/Hsp40/hdj-1), dj2 (DjA1/HSDJ/hdj-2), dj3 (DjA2), and dj4 (DjA4), in the liver of CCl4-treated rats was analyzed. Messenger ribonucleic acids for all these chaperones were markedly induced 3-12 hours after CCl4 treatment with a maximum at 6 hours. Hsp70 and dj1 proteins were markedly induced at 6-24 hours with a maximum at 12 hours, whereas dj2 and dj4 were moderately induced at around 12 hours. Hsc70 was weakly induced after treatment, and dj3 was little induced. To better understand the significance of the induction of chaperones, the effect of preinduction of chaperones on CCl4-induced liver damage was analyzed. When chaperones were preinduced in the liver by heat treatment, increase in serum alanine aminotransferase activity after CCl4 treatment was significantly attenuated. Hsp90, another major cytosolic chaperone, also was induced by heat treatment. On the other hand, Mn- and Cu/Zn-superoxide dismutase were not induced by heat treatment or by CCl4 treatment. These results suggest that cytosolic chaperones of Hsp70 and DnaJ families or Hsp90 (or both) are induced in CCl4-treated rat liver to protect the hepatocytes from the damage being inflicted.

Type I DnaJs comprise one type of Hsp70 cochaperones. Previously, we showed that two type I DnaJ cochaperones, DjA1 (HSDJ/Hdj-2/Rdj-1/dj2) and DjA2 (cpr3/DNAJ3/Rdj-2/dj3), are important for mitochondrial protein import and luciferase refolding. Another type I DnaJ homolog, DjA4 (mmDjA4/dj4), is highly expressed in heart and testis, and the coexpression of Hsp70 and DjA4 protects against heat stress-induced cell death. Here, we have studied the chaperone functions of DjA4 by assaying the refolding of chemically or thermally denatured luciferase, suppression of luciferase aggregation, and the ATPase of Hsp70s, and compared these activities with those of DjA2. DjA4 stimulates the hydrolysis of ATP by Hsp70. DjA2, but not DjA4, together with Hsp70 caused denatured luciferase to refold efficiently. Together with Hsp70, both DjA2 and DjA4 are efficient in suppressing luciferase aggregation. bag-1 further stimulates ATP hydrolysis and protein refolding by Hsp70 plus DjA2 but not by Hsp70 plus DjA4. Hsp70-2, a testis-specific Hsp70 family member, behaves very similarly to Hsp70 in all these assays. Thus, Hsp70 and Hsp70-2 have similar activities in vitro, and DjA2 and DjA4 can function as partner cochaperones of Hsp70 and Hsp70-2. However, DjA4 is not functionally equivalent in modulating Hsp70s.

DjB1 (Hsp40/DnajB1/Hdj1) is a member of the Hsp40/DnaJ family that functions as a co-chaperone of mammalian Hsp70s. DjB1 recognizes substrate proteins and facilitates the ATPase activity of Hsp70. We generated DjB1 deficient mice. The DjB1–/– mice were viable and fertile with no obvious abnormalities, thus indicating that DjB1 is dispensable for development and viability. No difference was found between the DjB1–/– and wild-type peritoneal macrophages regarding resistance against various types of apoptosis-inducing reagents. However, DjB1–/– cells showed decreased thermotolerance in the early phase after mild heat treatment, but not in the late phase. After the heat treatment, Hsp70 was induced similarly in wild-type and DjB1–/– cells. Immunofluorescence staining of wild-type cells revealed the accumulation of DjB1 and Hsc70 in the nucleus after heat treatment. DjB1 also accumulated in the centrosome. The accumulation of Hsc70 in the nucleus was also observed in DjB1–/– cells. These results suggest that the impaired thermotolerance of DjB1–/– cells is not due to a mislocation of the Hsp70 family. [ABSTRACT FROM PUBLISHER]

Most mitochondrial preproteins are maintained in a loosely folded import-competent conformation by cytosolic chaperones, and are imported into mitochondria by translocator complexes containing a preprotein receptor, termed translocase of the outer membrane of mitochondria (Tom) 20. Using two-hybrid screening, we identified arylhydrocarbon receptor-interacting protein (ALP), an FK506-binding protein homologue, interacting with Tom20. The extreme COOH-terminal acidic segment of Tom20 was required for interaction with tetratricopeptide repeats of AIP. An in vitro import assay indicated that AIP prevents preornithine transcarbamylase from the loss of import competency. In cultured cells, overexpression of AIP enhanced preornithine transcarbamylase import, and depletion of AIP by RNA interference impaired the import. An in vitro binding assay revealed that AIP specifically binds to mitochondrial preproteins. Formation of a ternary complex of Tom20, AIP, and preprotein was observed. Hsc70 was also found to bind to AIR An aggregation suppression assay indicated that AIP has a chaperone-like activity to prevent substrate proteins from aggregation. These results suggest that AIP functions as a cytosolic factor that mediates preprotein import into mitochondria. [ABSTRACT FROM AUTHOR]