Molecular chaperones facilitate the correct folding of other proteins under physiological and stress conditions. Recently it has become evident that various co-chaperone proteins regulate the cellular functions of these chaperones, particularly Hsp70 and Hsp90. Hop is one of the most extensively studied co-chaperones that is able to directly associate with both Hsp70 and Hsp90. The current dogma proposes that Hop functions primarily as an adaptor that directs Hsp90 to Hsp70-client protein complexes in the cytoplasm. However, recent evidence suggests that Hop can also modulate the chaperone activities of these Hsps, and that it is not dedicated to Hsp70 and Hsp90. While the co-chaperone function of Hop within the cytoplasm has been extensively studied, its association with nuclear complexes and prion proteins remains to be elucidated. This article will review the structural features of Hop, and the evidence that its biological function is considerably broader than previously envisaged.

The co-chaperone murine stress-inducible protein 1 (mSTI1), an Hsp70/Hsp90 organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. The mSTI1 protein can be phosphorylated in vitro by cell cycle kinases proximal to a putative nuclear localization signal (NLS), which substantiated a predicted casein kinase II (CKII)-cdc2 kinase-NLS (CcN) motif at position 180-239 and suggested that mSTI1 might move between the cytoplasm and the nucleus under certain cell cycle conditions. The mechanism responsible for the cellular localization of mSTI1 was probed using NIH3T3 fibroblasts to investigate the localization of endogenous mSTI1 and enhanced green fluorescent protein (EGFP)-tagged mSTI1 mutants. Localization studies on cell lines stably expressing NLSmSTI1-EGFP and EGFP demonstrated that the NLSmSTI1 was able to promote a nuclear localization of EGFP. The mSTI1 protein was exclusively cytoplasmic in most cells under normal conditions but was present in the nucleus of a subpopulation of cells and accumulated in the nucleus following inhibition of nuclear export (leptomycin B treatment). G1/S-phase arrest (using hydroxyurea) and inhibition of cdc2 kinase (using olomoucine) but not inhibition of casein kinase II (using 5,6-dichlorobenzimidazole riboside), increased the proportion of cells with endogenous mSTI1 nuclear staining. mSTI1-EGFP behaved identically to endogenous mSTI1. The functional importance of key residues was tested using modified mSTI1-EGFP proteins. Inactivation and phosphorylation mimicking of potential phosphorylation sites in mSTI1 altered the nuclear translocation. Mimicking of phosphorylation at the mSTI1 CKII phosphorylation site (S189E) promoted nuclear localization of mSTI1-EGFP. Mimicking phosphorylation at the cdc2 kinase phosphorylation site (T198E) promoted cytoplasmic localization of mSTI1-EGFP at the G1/S-phase transition,whereas removal of this site (T198A) promoted the nuclear localization of mSTI1-EGFP under the same conditions. These data provide the first evidence of nuclear import and export of a major Hsp70/Hsp90 co-chaperone and the regulation of this nuclear-cytoplasmic shuttling by cell cycle status and cell cycle kinases.

Self-renewal and differentiation of stem cells are tightly regulated processes subject to intrinsic and extrinsic signals. Molecular chaperones and co-chaperones, especially heat shock proteins (Hsp), are ubiquitous molecules involved in the modulation of protein conformational and complexation states. The function of Hsp, which are typically associated with stress response and tolerance, is well characterized in differentiated cells, while their role in stem cells remains unclear. It appears that embryonic stem cells exhibit increased stress tolerance and concomitant high levels of chaperone expression. This review critically evaluates stem cell research from a molecular chaperone perspective. Furthermore, we propose a model of chaperone-modulated self-renewal in mouse embryonic stem cells.

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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