Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp...

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Published in:	The international journal of biochemistry & cell biology Vol. 39; no. 4; pp. 736 - 751
Main Authors:	Nicoll, W.S., Botha, M., McNamara, C., Schlange, M., Pesce, E.-R., Boshoff, A., Ludewig, M.H., Zimmermann, R., Cheetham, M.E., Chapple, J.P., Blatch, G.L.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier Ltd 2007 Elsevier
Subjects:	Amino Acid Sequence Amino Acid Substitution Animals Base Sequence Binding Sites Cytosol - metabolism DnaJ Endoplasmic Reticulum - metabolism Endoplasmic reticulum Hsp40s Escherichia coli - genetics Escherichia coli - growth & development Escherichia coli - metabolism Escherichia coli Proteins - chemistry Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Genetic Complementation Test HSJ1 HSP40 Heat-Shock Proteins - genetics HSP40 Heat-Shock Proteins - metabolism HSP70 Heat-Shock Proteins - chemistry HSP70 Heat-Shock Proteins - genetics HSP70 Heat-Shock Proteins - metabolism Humans Malarial Hsp40s Mice Molecular Sequence Data Mutation Neoplasm Proteins - genetics Neoplasm Proteins - metabolism Phylogeny Protein Binding Protein Structure, Tertiary Protozoan Proteins - genetics Protozoan Proteins - metabolism Sequence Homology, Amino Acid Temperature Trypanosomal Hsp40s Malarial Hsp40s Endoplasmic reticulum Hsp40s DnaJ HSJ1 Trypanosomal Hsp40s
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Summary:	Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I–IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.
Bibliography:	These authors have contributed equally to this work.
ISSN:	1357-2725 1878-5875
DOI:	10.1016/j.biocel.2006.11.006