Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27‐kDa heat‐shock protein

The Drosophila melanogaster small heat-shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to a-crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. lho internal and adjacent deletion mutants in the conserved a-crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian-Virus-40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen-peroxide stresses as judged by Trypan-blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the Send of the conserved a-crystallin domain and covers the fnst 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N-terminal half of the conserved a-crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the a-crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus. The heat-shock or stress proteins (hsp) are synthesized at elevated temperatures or following perturbations of the cellular environment (reviewed in [l -31). These proteins are divided in several groups according to their molecular masses : hsp100-90, hsp70, hsp60 and the small hsp. In recent years, much information has emerged concerning the function of the high-molecular-mass hsp which appear to act as molecular chaperons. Hsp60 facilitates the correct folding of other proteins, particularly in the mitochondria [4]. Among its multiple functions, hsp70 binds to newly synthesized polypeptides and prevents their folding until appropriate interactions are established in the assembly of protein complexes [5, 61. Hsp90 was also shown to act as a protein chaperon and to associate with specific cellular proteins involved in signal transduction [7-91. The small hsp form a family of polypeptides with molecular masses in the range 20- 30 kDa. They are less conserved through evolution than the highmolecular mass hsp, despite the fact that they share a similarity with a region covering over 50% of the amino-acid sequence of both the bovine a-crystallin subunits A and B