Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Abstract

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp7O and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc7O, is especially enriched in embryos. hsc7O is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp7O-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp7O and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc7O have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.Drosophila melanogaster responds to growth at elevated temperature and a number of other stresses by suppressing normal protein synthesis and inducing or enhancing the synthesis of seven heat shock proteins (hsps), hsp70, -68, -83, -27, -26, -23, and -22 (4). The characteristic vigorous induction of a small number of evolutionarily conserved heat shock genes is now known to be a universal cellular response to stress. This response has been observed in a broad spectrum of eucaryotes, including plants, and also in the procaryote Escherichia coli (61). The mechanism of induction common to these inducers and the functions of the hsps are unknown. There is good evidence that the stress response is an adaptive mechanism that enables cells to survive a variety of environmental conditions that would otherwise be lethal (2,17,34,36,38).It is clear that the D. melanogaster heat shock genes are not coordinately expressed during normal development. hsp83 is probably present in all normal cells, and its induction after heat shock results in only a few fold increase in protein (33,40,76). mRNAs for hsp27 and -26 are synthesized during oogenesis and can be detected until the blastoderm stage (76). All four small hsps are induced in response to ecdysone during puparium formation (8, 25, 26, 58).In D. melanogaster, the Hsp7O gene family consists of both heat-inducible and constitutively expressed genes. hsp70 and hsp68 (which share 75% homology) are virtually absent under nonstress conditions (68). There are two copies of Hsp7O at 87A, three to four copies at 87C, and one copy of Hsp68 at 95D. Craig and colleagues (9, 23) have discovered a family of genes, 75 to 80% homologous to Hsp7O, termed heat shock cognate genes, which are expressed under normal growth conditions. The three heat shock cognate genes identified, Hscl, -2, and -4, map cytologically to 70C, 87D, and 88E, respectively. The levels of RNA organisms (20,28,35,71,72,74). A family of genes encoding proteins homologous to the Hsp7O constitutive and inducible members has also been identified and isolated in yeasts (24). In E. coli, heat treatment stimulates the rates of synthesis of at least 17 proteins (39), of which DnaK and C62.5 have been shown to be homologous to hsp70 and hsp83, respectively, in D. me...

Two missense alleles of the Drosophila melanogaster act88F actin gene are strongly antimorphic but only weakly induce synthesis of heat shock proteins.

“…6) appeared to be greater in the three mutant genotypes than in the Canton-S strain. This latter result supports recent data of Palter et al (22), who found that levels of Hsp7O-cognate mRNA increased twofold after exposure to heat. Presumably, a stress situation caused by a defective actin isoform could result in a comparable level of HSP-cognate protein induction.…”

“…(27). After blocking for 1 h in 50 mM Tris (pH 7.4)-3% bovine serum albumin-0.9% NaCl-0.04% sodium azide, blots were successively incubated for 3 h with a 1:100 dilution of monoclonal antibody IPC4 (22) (18). In all three cases the mutation causing flightless phenotype maps to a chromosomal position coincident with the location of the act88F gene.…”

“…The thoracic proteins of normal and heat-shocked wild-type flies and of each mutant homozygote were separated on two-dimensional gels and blotted to nitrocellulose. The filters were subsequently reacted with IPC4, a monoclonal antibody which recognizes Hsp7O and cognate proteins (22). Bound IPC4 was visualized by reaction with a peroxidaseconjugated second antibody.…”

Heat shock proteins: the search for functions.

“…The HSP70 gene itself is not activated under this condition but proteins for two HSPT0 family members are induced (35). An HSP70-1ike protein is one of the first proteins synthesized after zygote formation in mouse embryogenesis (4), and several HSPs are induced during oogenesis and pupation in Drosophila (25,36,44,59,70). …”

“…Three of these are known only by their subunit molecular weights: HSP90, HSP70, and HSP 20-30. The precise molecular weight varies slightly among different organisms, In the eukaryotic cell, each group appears to constitute a gene family consisting of closely related protein isoforms and genes encoding sequences regulated by different promoters (14,16,19,46,50,59,79). The fourth HSP is ubiquitin, a protein of 8,000 mol wt.…”