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The Hsp70-Hsp90 complex is implicated in the folding and regulation of numerous signaling proteins, and Hop, the Hsp70-Hsp90 Organizing Protein, facilitates the association of this multichaperone machinery. Phosphatase treatment of mouse cell extracts reduced the number of Hop isoforms compared to untreated extracts, providing the first direct evidence that Hop was phosphorylated in vivo. Furthermore, surface plasmon resonance (SPR) spectroscopy showed that a cdc2 kinase phosphorylation mimic of Hop had reduced affinity for Hsp90 binding. Hop was predominantly cytoplasmic, but translocated to the nucleus in response to heat shock. A putative bipartite nuclear localization signal (NLS) has been identified within the Hsp90-binding domain of Hop. Although substitution of residues within the major arm of this proposed NLS abolished Hop-Hsp90 interaction as determined by SPR, this was not sufficient to prevent the nuclear accumulation of Hop under leptomycin-B treatment and heat shock conditions. These results showed for the first time that the subcellular localization of Hop was stress regulated and that the major arm of the putative NLS was not directly important for nuclear translocation but was critical for Hop-Hsp90 association in vitro. We propose a model in which the association of Hop with Hsp90 and the phosphorylated status of Hop both play a role in the mechanism of nucleo-cytoplasmic shuttling of Hop.

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Stress responses can be measured at various levels of biological organization, from sub-organism through to ecosystem level. This study aimed to investigate stress protein induction as a sub-organism level stress response in two South African freshwater aquatic macroinvertebrates exposed to detergent linear alkylbenzene sulfonate (LAS). Shrimp Caridina nilotica and limpet Burnupia stenochorias were exposed to a range of LAS concentrations (0, 1, 1.8, 3.2, 6.5 and 12.7 mg/L and 0, 0.6, 1.2, 1.6 and 3.2 mg/L respectively) for 96 h. Surviving organisms were prepared for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis. In C. nilotica there appeared to be induction of a putative ≈ 70 kDa protein at 12.7 mg/L LAS, and induction of putative ≈45 and ≈40 kDa proteins at concentrations of 6.5 mg/L LAS and above. However, only an Hsp70 protein was detected with anti-Hsp72/Hsc73 at 12.7 mg/L LAS. No protein induction was observed in exposed B. stenochorias, however an Hsp40 protein was detected with anti-Hsp40 in exposed and unexposed limpets.

The role of the TPR2B domain of Hop is as yet unknown. We have shown here by site directed mutagenesis and size exclusion chromatography for the first time that the TPR1 and TPR2B domains of Hop independently dimerized, and that the dimerization of TPR2B was not dependent on its predicted two-carboxylate clamp residues. Furthermore, our data indicated that the dimerization of Hop and its domains was not disrupted in the presence of Hsp70 and Hsp90 peptides.

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