DnaJ (Hsp40) is the J-domain co-chaperone of the DnaK (Hsp70) chaperone system. It binds unfolded and misfolded substrate proteins and, through its N-terminal J domain, stimulates the ATPase activity of DnaK, driving the formation of a stable DnaK-substrate complex; the nucleotide-exchange factor GrpE then releases ADP from DnaK and ATP rebinding releases the substrate, completing an iterative folding cycle. DnaJ also has an autonomous, DnaK-independent holdase/chaperone activity that prevents the aggregation of stress-denatured proteins and helps disaggregate them. It is a cytoplasmic homodimer that coordinates two structural zinc ions per monomer through a cysteine-rich (CR-type) zinc-finger domain; zinc center 1 supports the autonomous chaperone activity and zinc center 2 is required for interaction with DnaK. Together with DnaK and GrpE, DnaJ mediates the cellular response to heat and hyperosmotic stress, promotes protein folding, refolding and disaggregation, and participates in the activation of replication-initiation proteins during plasmid and phage DNA replication.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005524 ATP binding | IEA GO_REF:0000002 | REMOVE | Summary: This annotation is incorrect. DnaJ does not itself bind or hydrolyze ATP; it is a co-chaperone that stimulates the ATPase activity of its partner DnaK (Hsp70). It is DnaK, not DnaJ, that binds and hydrolyzes ATP. The UniProt record lists no ATP-binding site or feature for DnaJ, and there is no Walker motif in the sequence. This is a known erroneous InterPro2GO propagation to the DnaJ family and should be removed. Reason: DnaJ has no ATP-binding site or Walker motif in its sequence; ATP binding and hydrolysis are properties of its partner DnaK, so this is an erroneous family-level InterPro2GO transfer. Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt with the DnaJ-bound protein, DnaK hydrolyzes its bound ATP, resulting file:PSEPK/dnaJ/dnaJ-notes.md DnaJ does NOT itself bind/hydrolyze ATP. It stimulates the ATPase activity of DnaK; the ATP-binding partner is DnaK. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Correct subcellular localization. UniProt assigns DnaJ to the cytoplasm, where it acts together with DnaK and GrpE on cytoplasmic substrate proteins. Reason: The curated UniProt subcellular location places DnaJ in the cytoplasm, consistent with its action on cytoplasmic substrate proteins. Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0006457 protein folding | IEA GO_REF:0000002 | ACCEPT | Summary: Correct and a core biological process. As the J-domain co-chaperone of the DnaK system, DnaJ delivers unfolded substrates to DnaK and drives iterative ATP-dependent folding cycles, and it also has autonomous chaperone activity that prevents aggregation of stress-denatured proteins. Reason: Protein folding is a direct, core biological process for this J-domain co-chaperone of the DnaK folding cycle. Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt interactions between DnaJ, DnaK and GrpE are required for fully |
| GO:0008270 zinc ion binding | IEA GO_REF:0000104 | ACCEPT | Summary: Correct and specific. DnaJ binds two structural Zn(2+) ions per monomer through its cysteine-rich (CR-type) zinc-finger domain (eight coordinating Cys residues). Zinc center 1 supports the autonomous chaperone activity and zinc center 2 is required for interaction with DnaK. This is a specific metal-ion term (no broad 'metal ion binding' annotation is present in this GOA to down-rank). Reason: Zinc binding is specific and structurally essential (two CR-type zinc centers required for chaperone activity and DnaK interaction); no broader metal-ion term is present to supersede it. Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt Binds 2 Zn(2+) ions per monomer. file:PSEPK/dnaJ/dnaJ-notes.md Zinc center 1 plays an important role in the autonomous, DnaK-independent chaperone activity of DnaJ. Zinc center 2 is essential for interaction with DnaK and for DnaJ activity. |
| GO:0009408 response to heat | IEA GO_REF:0000002 | ACCEPT | Summary: Correct and a core biological process. DnaJ participates actively in the response to heat shock (and hyperosmotic stress) by preventing the aggregation of stress-denatured proteins and disaggregating them, as part of the DnaK/DnaJ/GrpE heat-shock chaperone machinery. Reason: DnaJ is a core component of the DnaK/DnaJ/GrpE heat-shock machinery that prevents and reverses heat-induced protein aggregation. Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt heat shock by preventing the aggregation of stress-denatured proteins |
| GO:0031072 heat shock protein binding | IEA GO_REF:0000002 | MODIFY | Summary: The essence is correct - DnaJ physically binds DnaK, which is an Hsp70 heat-shock protein. However, 'heat shock protein binding' is imprecise; the more informative term 'protein-folding chaperone binding' (GO:0051087) better captures the defining co-chaperone interaction with the DnaK chaperone that DnaJ stimulates. Reason: The interaction is real but 'heat shock protein binding' is imprecise; 'protein-folding chaperone binding' specifically captures the defining DnaK (Hsp70) co-chaperone interaction that DnaJ stimulates. Proposed replacements: protein-folding chaperone binding Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt with the DnaJ-bound protein, DnaK hydrolyzes its bound ATP, resulting file:PSEPK/dnaJ/dnaJ-uniprot.txt is essential for interaction with DnaK and for DnaJ activity |
| GO:0042026 protein refolding | IEA GO_REF:0000118 | ACCEPT | Summary: Correct and a core biological process. Through repeated ATP-dependent cycles with DnaK and GrpE, and via its autonomous disaggregation activity, DnaJ promotes the refolding of denatured proteins to the native state. Reason: Refolding of denatured proteins, via DnaK-coupled ATP-dependent cycles and autonomous disaggregation, is a direct, core biological process for DnaJ. Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt and by disaggregating proteins, also in an autonomous, DnaK-independent |
| GO:0001671 ATPase activator activity | IEA GO_REF:0000002 | NEW | Summary: Proposed new core annotation. DnaJ's defining molecular function is not captured by the current GOA. Via its J domain, DnaJ binds to and stimulates the intrinsic ATPase activity of the DnaK (Hsp70) chaperone, driving stable substrate capture. This ATPase activator activity is annotated to Hsp40 proteins in Reactome (HSP40s activate intrinsic ATPase activity of HSP70s) and is the most informative molecular function for this co-chaperone. Reason: ATPase activator activity is the defining, most informative molecular function of this J-domain co-chaperone and is missing from the current GOA. Supporting Evidence: file:PSEPK/dnaJ/dnaJ-uniprot.txt The J domain is necessary and sufficient to stimulate DnaK file:PSEPK/dnaJ/dnaJ-uniprot.txt PANTHER; PTHR43096:SF48; CHAPERONE PROTEIN DNAJ |
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Download this section (compressed HTML)Q: Does the autonomous, DnaK-independent holdase/disaggregation activity of DnaJ contribute measurably to proteostasis and stress survival in Pseudomonas putida, or is essentially all of its in vivo function mediated through DnaK?
Q: What is the substrate spectrum of DnaJ in P. putida under the solvent, oxidative, and heat stresses relevant to its environmental and biotechnological niche, and how does it compare with the well-characterized E. coli ortholog?
Q: How is dnaJ expression coordinated with the rpoH/sigma-32 heat-shock regulon and with dnaK/grpE in P. putida, and is it co-transcribed in a conserved operon?
Experiment: Construct a dnaJ deletion mutant in P. putida KT2440 and assay growth and viability after heat shock and hyperosmotic stress, alongside dnaK and grpE mutants, to test the requirement of the J-domain co-chaperone for stress tolerance and proteostasis.
Type: Gene knockout and stress survival assay
Experiment: Purify recombinant P. putida DnaJ, DnaK, and GrpE and measure DnaJ-dependent stimulation of DnaK ATPase activity and reactivation of a heat-denatured model substrate (e.g. luciferase or malate dehydrogenase), including J-domain HPD-motif point mutants to confirm the mechanism.
Type: In vitro ATPase stimulation and refolding assay
Experiment: Mutate the cysteine residues of zinc center 1 versus zinc center 2 and assay the effects on autonomous chaperone (anti-aggregation) activity versus DnaK interaction, to dissect the distinct roles of the two zinc sites in P. putida DnaJ.
Type: Zinc-center mutagenesis and functional assay
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