The Chaperoning Activity of hsp110

Abstract

hsp110 is one of major heat shock proteins of eukaryotic cells and is a diverged relative of the hsp70 family. It has been previously shown that hsp110 maintains heat-denatured luciferase in a soluble, folding competent state and also confers cellular heat resistance in vivo. In the present study the functional domains of hsp110 that are responsible for its chaperoning activity are identified by targeted deletion mutagenesis using the DnaK structure as the model. The chaperoning activity of mutants is assessed based on their ability to solubilize heat-denatured luciferase as well as to refold luciferase in the presence of rabbit reticulocyte lysate. It is shown that these functions require only an internal region of hsp110 that includes the predicted peptide binding domain and two immediately adjacent C-terminal domains. It is also shown that although hsp110 binds ATP, binding can be blocked by its C-terminal region. hsp701 is perhaps the best studied of the major heat shock proteins. hsp70 family members have been shown to play essential roles in a variety of cellular activities, for example, folding of nascent polypeptides, protein translocation across intracellular membranes, and regulation of the activities of steroid hormone receptors and kinases (1-3). The unifying mechanism for their action is based on their chaperoning activity, i.e. their ability to recognize and bind to peptide segments that are not normally exposed to the aqueous environment because they are normally buried in the interior of the protein or are hidden by interactions with other proteins (4 -6). Multiple members of hsp70 family occur within individual organisms, and it is believed that these different hsp70s perform differing roles. In Saccharomyces cerevisiae, it has been shown that different hsp70 family members are required for specific functions, e.g. transport across membranes or translation, and that one member may not be interchanged for another (7)(8)(9). This division of labor occurs despite the very high degree of amino acid sequence identity between these hsp70 family members, usually better than 60%.Based on strong inducibility, quantity, and presence in many cell types, hsp110 has also been recognized for the last two decades as a major heat shock protein, specifically in mammalian cells (10 -13). hsp110 has been recently cloned from a variety of organisms as diverse as yeast and man (14 -23). Surprisingly, the cloning of hsp110 family members has indicated that this family does not represent a genetically unique stress protein group, as previously seen with other heat shock protein families such as hsp90 or 28, but that they are clearly related to the hsp70 family (14). The hsp110s in S. cerevisiae have been termed the Stress Seventy E (SSE) family (23). However, the hsp110 family is a distinct subset of the hsp70 family which, in addition to their significantly increased mass compared with the hsp70s, differ in their significant sequence divergence from the archetypical hsp70s (14). In light of the differential functions...

The Function of the Yeast Molecular Chaperone Sse1 Is Mechanistically Distinct from the Closely Related Hsp70 Family

“…Hsp110 and Grp170 proteins are significantly larger than Hsp70, possessing a loop region between the ␤-and ␣-subdomains, as well as an extended tail. Hsp110 has been shown to bind denatured proteins in vitro but is unable to actively refold them and instead acts as a "holdase," maintaining substrate polypeptides in a folding-competent state (9,10). No endogenous substrates have been identified for the Hsp110s to date; however overexpression of Hsp110 increases thermotolerance in Chinese hamster ovary (CHO) cells (9).…”

Hsp110 Cooperates with Different Cytosolic HSP70 Systems in a Pathway for de Novo Folding

“…2, 6, and 7). A unique subclass of Hsp70s called Hsp110s, found only in eukaryotic cells, is distinguished by an insertion of flexible loops after the polypeptide binding domain and an extended tail at the C terminus (8,9). The function of these additional domains in Hsp110 remains unclear.…”

The Yeast Hsp110 Sse1 Functionally Interacts with the Hsp70 Chaperones Ssa and Ssb

“…The defining characteristics of the Hsp110 family are an extended linker region (ϳ98 residues versus ϳ9 in Hsp70) separating the ␤-sandwich peptide binding fold from the ␣-helical lid domain, and an extended C-terminal tail region whose structure and function is unknown (5). Importantly, Hsp110 cannot actively refold proteins but is able to hold thermally denatured model substrates such as luciferase in a protected state such that they can be more efficiently renatured by folding-competent chaperones such as Hsp70 (21). Because of this property, Hsp110 is classified as a "holdase," similar to Hsp90 and many of its co-chaperones, rather than a "foldase."…”