A major mammalian heat shock protein of 110 kDa (hsp110) has long been observed, but has not been cloned. We have cloned the hamster cDNA for hsp110 and show that it hybridizes on a Northern blot to a 3.5-kilobase heat-inducible message in hamster and mouse. The hsp110 sequence was found to share an approximately 30-33% amino acid identity with members of the hsp70 family, most of which occurs in the conserved ATP-binding domain of these molecules. In addition, five sequences were found to be highly similar to hsp110. These are the sea urchin egg receptor for sperm (Foltz, K.R., Partin, J. S., and Lennarz, W.J. (1993) Science 259, 1421-1425) and additional sequences from human and Caenorhaditis elegans and two from yeast. The carboxyl-terminal two-thirds of hsp110 and these five related proteins contain a pattern of highly conserved regions of sequence unique to this group. A probe containing these conserved sequences was found to strongly cross-react on a Southern blot with genomic sequences from yeast to man. A Western blot analysis of several murine tissues indicates that hsp110 is constitutively expressed in all mouse tissues and is highly expressed in brain. Therefore, hsp110 belongs to a new category of large and structurally unique stress proteins that are the most distantly related known members of the hsp70 family.

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...