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 alpha-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. Two internal and adjacent deletion mutants in the conserved alpha-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 5' end of the conserved alpha-crystallin domain and covers the first 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 alpha-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 alpha-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.
Mehlen
P
P
Molecular and Cellular Genetics, CNRS UMR-106, Claude Bernard University, Lyon, France.
Briolay
J
J
Smith
L
L
Diaz-latoud
C
C
Fabre
N
N
Pauli
D
D
Arrigo
A P
AP
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002470:Cell Survival; D005591:Chemical Fractionation; D002522:Chlorocebus aethiops; D003459:Crystallins / Q000737:chemistry; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006360:Heat-Shock Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007668:Kidney; D009154:Mutation; D014162:Transfection / Q000235:genetics; D014343:Trypan Blue
D003459:Crystallins; D006360:Heat-Shock Proteins; D006861:Hydrogen Peroxide; D014343:Trypan Blue