Cooperative interaction of Hsp40 and TPR1 with Hsp70 reverses Hsp70-HspBp1 complex formation.
Hsp40 and TPR1 are chaperone adaptors that regulate Hsp70-dependent folding processes by interacting with the amino terminal and carboxy terminal domains of Hsp70, respectively. In this study, we report cooperative interactions involving Hsp70, Hsp40, and TPR1 that enhance Hsp70-dependent folding of chemically denatured substrates. Hsp40 and Hsp70 dependent folding of chemically denatured luciferase was enhanced by up to 80% when TPR1 was also present. HspBp1, a negative modulator of Hsp70, completely inhibited Hsp70-dependent folding in the presence of Hsp40. However, when TPR1 was included in the reaction, the inhibitory effect of HspBp1 was reversed. To analyze the interactions, Kd analysis and competition assays were carried out. The Kds of the interactions of Hsp40, TRP1, and HspBp1 with Hsp70 were 0.5, 0.6, and 0.04 mM, respectively. Interestingly, the Hsp70/HspBp1 complex could only be dissociated in the presence of both Hsp40 and TPR1, suggesting cooperative interaction between Hsp70, Hsp40 and TPR1. To examine these interactions in vivo, we established a tetracycline-regulatable Hela cell line that expresses Hsp70 in the absence of doxycycline. Expression of HspBp1 inhibited Hsp70-dependent folding of heat-denatured luciferase, and this effect was only reversed in the presence of Hsp40 and TPR1. Our findings reveal a novel mechanism of positive regulation of Hsp70-dependent folding.
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D006801:Humans; D046911:Macromolecular Substances; D017510:Protein Folding
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; C494647:DNAJB1 protein, human; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C116331:HSPBP1 protein, human; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; C475144:TTC1 protein, human; D020962:Heterotrimeric GTP-Binding Proteins