In response to low ambient temperature, mammalian cells as well as microorganisms change various physiological functions, but the molecular mechanisms underlying these adaptations are just beginning to be understood. We report here the isolation of a mouse cold-inducible RNA-binding protein (cirp) cDNA and investigation of its role in cold-stress response of mammalian cells. The cirp cDNA encoded an 18-kD protein consisting of an amino-terminal RNAbinding domain and a carboxyl-terminal glycine-rich domain and exhibited structural similarity to a class of stress-induced RNA-binding proteins found in plants. Immunofluorescence microscopy showed that CIRP was localized in the nucleoplasm of BALB/3T3 mouse fibroblasts. When the culture temperature was lowered from 37 to 32°C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37°C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37°C with prolongation of G1 phase of the cell cycle. These results indicate that CIRP plays an essential role in cold-induced growth suppression of mouse fibroblasts. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in mammalian cells.

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We isolated a novel hsp110-related gene, apg-1, from a testis cDNA library. The apg-1 transcripts were constitutively expressed in the testicular germ cells and, in some degree, most tissues examined. In a mouse TAMA26 Sertoli cell line, apg-1 transcripts were induced in 2 h by a temperature shift from 32 to 39°C, but not by a shift from 37 to 42°C, the traditional heat stress, or a shift from 32 to 42°C. The heat response pattern of hsp110 expression was similar to that of apg-1. Although induction of a hsp70 transcript was observed in 2 h by a shift from 32 to 39°C, the induction was more apparent by a shift from 37 to 42°C or from 32 to 42°C. Essentially similar differential response patterns were observed among these genes in NIH/3T3 fibroblasts as well. The nuclear run-on assay and the native gel mobility shift assay demonstrated that, by the 32 to 39°C temperature shift, the apg-1 gene was transcriptionally activated, and heat shock factor 1 bound to the heat shock elements in the 5 -flanking region of the apg-1 gene. These results demonstrated that expressions of apg-1, hsp110, and hsp70 could be heat-induced at a temperature lower than the traditional elevated temperatures in somatic cells of both testis and nontestis origin and suggest that the mechanisms regulating the transcript levels of apg-1 and hsp110 are different from those of hsp70. Furthermore, the constitutive expression in germ cells suggests that APG-1 plays a specific role in spermatogenesis as well as in stress response.Prokaryotic and eukaryotic organisms respond to elevated temperatures by synthesizing a distinct set of proteins termed heat shock proteins (HSPs) 1 (1). Anoxia, ethanol, radiation, inflammation, and certain heavy metal ions also induce HSPs in the cells. An early and long-standing assumption regarding the heat shock response was that the HSPs protected cells from the toxic effects of heat and other stresses. Subsequently, a series of studies revealed that HSPs are also present in cells at normal temperatures. Now members of the HSP family are established as molecular chaperones, assisting in the folding and unfolding, assembly and disassembly, and transport of various proteins (2-5). HSPs are also shown to interact with mutant p53 and p60 v-src (6 -8), suggesting their involvement in cell cycle regulation.Spermatogenesis begins at puberty and consists of three steps: the mitotic proliferation of the spermatogonia, meiosis at the spermatocyte stage, and the distinct cellular structural changes of the spermatids. Unlike somatic cells, the male germ cells are easily damaged at the body cavity temperature (9), indicating the presence of a differential heat sensitivity between somatic cells and germ cells. Sarge (10) recently reported that the temperature threshold for induction of HSP72 encoded by the hsp70 gene was lower in male germ cells than in somatic cells. To date, several HSPs have been found to be constitutively expressed in germ cells at specific stages of development. Two HSP70-related genes, hsp70.2 and hsc70t, ar...

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...