Role of the J-domain in the cooperation of Hsp40 with Hsp70

Abstract

The Genetic and biochemical evidence suggests that DnaJ functionally and physically interacts with DnaK even though no stable binary complex of these two proteins has been observed. DnaJ and DnaK cooperate in many cellular processes, including DNA replication (1-3), protein export (4), and stress response (5, 6). DnaJ and DnaK promote folding of denatured or partially unfolded proteins (7, 8) as well as newly synthesized polypeptides (9). DnaJ can act as a chaperone on its own (6, 8), or it can work together with DnaK (10). DnaK possesses a weak intrinsic ATPase activity (2), and ATP binding and hydrolysis are associated with conformational changes in DnaK that regulate substrate binding and release (11,12). DnaJ stimulates ATP hydrolysis by DnaK (13), and DnaK (MgADP) has the highest affinity toward polypeptide substrates (14, 15).The interaction between DnaJ and DnaK appears to be bimodal. A truncated DnaJ molecule consisting of the Jdomain and Gly͞Phe region stimulates the ATPase activity of DnaK and supports bacteriophage DNA replication, although a higher concentration of the truncated protein is required (16,17). The J-domain by itself neither stimulates the ATPase activity of DnaK nor supports replication of bacteriophage DNA. However, the J-domain plus a peptide added in trans act synergistically to stimulate DnaK's ATPase activity 200-fold (17).Point mutations in the J-domains of DnaJ and DnaJ homologs abolish the ability of these proteins to function with Hsp70 proteins. These mutations include H33Q in DnaJ (4, 16) and two mutations (18) of sec63p, P156N and D157A, which substitute residues in the conserved tripeptide HPD of the J-domain. An insertion of one amino acid between P34 and D35 in DnaJ also abolishes activity in a bacteriophage DNA replication assay (19). Because the tripeptide has no obvious structural role (20) and because there is a high degree of conservation in the HPD segment, the HPD tripeptide could mediate specific interactions between Hsp40 and Hsp70 proteins.The J-domain-Hsp70 interaction can be subverted by viruses. The J-domain of polyomavirus T antigens is required for viral DNA replication and tissue transformation, possibly by mediating interactions with Hsp70. Simian virus 40 (SV40) large T antigen associates with the constitutively expressed Hsc70 (21), and certain J-domain mutants defective in tissue transformation fail to bind Hsc70 (22). Chimeras constructed by substituting the J-domain of DnaJ with the J-domain from any of several polyomavirus T antigens can replace wild-type DnaJ in vivo (23). Likewise, chimeras of T antigen containing the J-domain from DnaJ or a eukaryotic Hsp40 support SV40 DNA replication (24). Nevertheless, the role of the T antigen J-domain in viral DNA replication has not been elucidated.C-terminal domains of DnaJ-family proteins may confer specificity for substrates and͞or function. The C-terminal domains are less well-conserved than the J-domain (25). Most, but not all, DnaJ homologs contain a domain that binds two Zn 2ϩ atoms, a featur...

The Presequence Translocase-associated Protein Import Motor of Mitochondria

“…Inclusion of the HPD Motif in Pam16 Does Not Alter Its Interaction with and Inhibition of Pam18 -The strongly conserved HPD sequence motif in the center of a J-domain (33) that is required for the interaction with Hsp70 and the stimulation of the ATPase activity is missing in Pam16. We asked if the generation of an HPD motif could convert Pam16 to a protein with an activity similar to other proteins of the DnaJ family, i.e.…”

“…Pam18 and Pam16 form a stable subcomplex that associates with the presequence translocase and the other motor subunits (31,32). However, Pam18 contains a J-domain that is responsible for the interaction with Hsp70 and is con-served in all proteins of the J-protein family (33)(34)(35)(36), whereas Pam16 contains a J-related domain only. The Pam16 domain possesses a similar length and predicted secondary structure as the Pam18 domain, but the characteristic sequence motif HPD (His-Pro-Asp) that is strictly conserved in all known Jproteins is missing in Pam16.…”

The in vivo function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain

“…A polypeptide first interacts with an Hsp70 in the ATP-bound state, then hydrolysis of ATP to ADP stabilizes this interaction, which is subsequently destabilized by the exchange of ADP for ATP. This cycle of interaction of Hsp70s with unfolded proteins is facilitated by J-type chaperones, which contain a canonical J domain that interacts with the ATPase domain of Hsp70s (12). In addition, some, but not all, J-type proteins bind unfolded or partially folded polypeptides, preventing their aggregation, and targeting them to Hsp70s (13).…”

DjlA Is a Third DnaK Co-chaperone of Escherichia coli, and DjlA-mediated Induction of Colanic Acid Capsule Requires DjlA-DnaK Interaction

“…Indeed, the J-domain of DnaJ is absolutely essential for its interaction with DnaK and is specifically required for stimulation of the ATPase activity (1,(7)(8)(9). A highly conserved HPD tripeptide, located in an exposed loop of the J-domain, is critical for co-chaperone function because mutations in these residues severely compromise the stimulation of ATP hydrolysis, not only in E. coli, but in many other organisms (5,10,11).…”