The Human DnaJ Homologue dj2 Facilitates Mitochondrial Protein Import and Luciferase Refolding

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.

The Endoplasmic Reticulum Chaperone Cosmc Directly Promotes in Vitro Folding of T-synthase

“…Importantly, addition of the general ER chaperone BiP did not restore the activity of heat-denatured T-synthase, whereas BiP does support renaturation of luciferase. It has been shown previously that recombinant Hsc70 can support in vitro refolding of heat-denatured luciferase (38). Furthermore, 6ϫHis-msCosmc E152K cannot restore the denatured HPC4-sT-syn in vitro as efficiently as 6ϫHis-sCosmc and 6ϫHis-sCosmc does not effect restoration of activity for another well-characterized ␤-galactosyltransferase (␤4-GalT).…”

hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria

“…Polyclonal anti-human dj1 and dj2 antibodies were those described elsewhere. 28 Monoclonal antibody against mouse iNOS was obtained from Transduction Laboratories (Lexington, KY, USA). A polyclonal antibody against mouse CHOP and polyclonal and monoclonal antibodies against human Bax were obtained from Santa Cruz Biotechnology Inc. Polyclonal antibody against GFP was as described elsewhere.…”

“…25,26 Several mammalian DnaJ homologs (hsp40 family members) have been identified, 27 and it seems likely that hsp70 family members are regulated by these homologs. 28 Major DnaJ homologs in mammalian cytosol are dj1 (DNAJB1: hsp40/ hdj-1) and dj2 (DNAJA1: HSDJ/hdj-2). We reported that dj1-hsp70 and dj2-hsp70 chaperone pairs are both effective in preventing NO-mediated apoptosis upstream of both caspase 3 activation and cytochrome c release from the mitochondria.…”

Multiple 40-kDa Heat-Shock Protein Chaperones Function in Tom70-dependent Mitochondrial Import

“…A polypeptide substrate commonly used to study Hsc70 function is firefly luciferase, which has a sensitive and reproducible enzymatic assay. Purified Hsc70 has been shown to assist luciferase refolding in numerous studies (Hohfeld et al, 1995;Minami et al, 1996;Hohfeld and Jentsch, 1997;Terada et al, 1997;Luders et al, 2000;Terada and Mori, 2000). However, in most cases the stress-inducible cochaperone Hsp40 was used, and a direct comparison between all three DJAs with Hsc70 has not yet been reported.…”