Highly conserved glucose-regulated protein in hamster and chicken cells: preliminary characterization of its cDNA clone.

Abstract

A temperature-sensitive mutant K12 derived from a Chinese hamster fibroblast has been shown to overproduce three specific proteins of Mr 94,000, 78,000, and 58,000 when incubated at the nonpermissive temperature (40.50C). We previously identified these proteins as glucose-regulated proteins similar to those observed in chicken embryo fibroblasts when the cells are starved of glucose. In this report, we show that the Mr 78,000 proteins isolated from the hamster K12 cell line and from chicken embryo fibroblasts have identical electrophoretic mobilities in two-dimensional isoelectric focusing gels and nearly identical peptide maps. However, these proteins are different from heat-shock proteins previously described for animal cells. We have constructed a library ofcDNA clones by using the RNA extracted from the hamster K12 cells incubated at 40.50C. Clones that hybridize preferentially with cDNA made from RNA at 40.50C were selected. By using the hybrid-selection technique, followed by in vitro translation, a cDNA clone containing a 2550-nucleotide insert coding for the hamster Mr 78,000 protein has been identified.It has been observed in various animal cell lines that, when the glucose in the growth medium is depleted, the synthesis of a specific set ofproteins is enhanced (1-4). Two of these proteins observed in chicken embryo fibroblasts have Mrs of 90,000-95,000 and 73,000-79,000 and were originally thought to be transformation-specific proteins (5, 6). Subsequently, it was demonstrated that increased contents of these proteins were actually due to rapid depletion of glucose from the growth medium by the transformed cells (1). The accumulation of three specific proteins (Mr 94,000, 78,000, and 58,000) related to the glucose-regulated proteins in a temperature-sensitive hamster mutant cell line, K12, has been reported (4). This cell line is particularly useful for the study of the coordinated expression ofthese proteins because, when the cells are shifted to the nonpermissive temperature (40.50C), the increase in synthesis of the glucose-regulated proteins is 5-fold to 10-fold greater than that obtained by glucose starvation. This accumulation is primarily due to new transcription and synthesis (7). Thus, the hamster K12 cells provide a convenient system for the molecular cloning of the genes coding for these proteins.In this report we describe experiments which characterize the relationship of the Mr 78,000 glucose-regulated proteins produced by hamster and chicken embryo fibroblasts. Our results indicate that this protein is highly conserved in the two species. Contrary to an earlier speculation (8), we demonstrate that the glucose-regulated proteins are different from heatshock proteins observed in animal cells. We also present preliminary evidence for the identification ofa cDNA clone coding for the Mr 78,000 glucose-regulated protein.MATERIALS AND METHODS Cell Lines and Culture Conditions. Conditions for culturing the hamster cell lines and labeling the cellular proteins have been described (4). Fo...

The protein disulphide-isomerase family: unravelling a string of folds

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Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes.

“…The Chinese hamster fibroblast cell lines WglA and K12 have been described previously (9,17). K12 GRP94, respectively, has been described previously (11,12). The pSVOCAT plasmid was provided by B. Howard.…”

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