[
J Mol Biol,
2009]
Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.
[
East Asia Worm Meeting,
2004]
Environmental and physiological stresses such as heat shock, oxidative stress, heavy metals and pathogenic conditions induce the cellular stress response. This response is often related to the heat shock proteins (HSPs) that function as molecular chaperones or proteases. Stress-inducible phosphoprotein (STI1) was first described in Saccharomyces cerevisiae , where it was implicated in mediating the heat shock response. However, the biological role of STI1 in vivo is poorly understood. We are using C. elegans to characterize the function of CeSTI-1 under stress conditions. The C. elegans gene R09E12.3 (LG V) is predicted to be a STI1 homologue that shows approximately 56% amino acids identity to human stress induced phosphoprotein 1. The CeSTI-1 is expressed from larva to adult in the pharynx, intestine, nerve cord and male tail. We have recently isolated a deletion mutant of Cesti-1 by PCR based TMP-UV mutagenesis. The Cesti-1 mutant contains a 917bp deletion from the upstream of the first exon to the part of fourth exon. This mutant shows decreased brood size and short life span under heat shock condition, suggesting that CeSTI-1 is related to stress response. Currently, in order to understand how Cesti-1 is regulated, we are analyzing the Cesti-1 promoter by mutation analysis. A search for the putative heat shock element (HSE) (TTCTa/cGAA) and DAF-16 binding element (DBE) (Ta/gTTTAC) in the sequence of Cesti-1 yields two HSE and two DBE at upstream from the ATG. We predicted that the Cesti-1 HSE and DBE may be a direct target of HSF-1 and DAF-16. To determine whether Cesti-1 is a direct target of HSF-1 and DAF-16, we are making several recombinant Cesti-1 promoter GFP translational constructs. In addition, STI1 proteins from several species have been described to make complexes with stress proteins of the HSP70 and HSP90 families. To investigate whether CeSTI-1 makes complex with HSPs like other species, we are going to perform in vitro binding assay with GST-HSPs and CeSTI-1.