Common among all organisms is an essential, highly conserved and exquisitely regulated cellular response to stressful environments. The heat shock response represents an adaptative mechanism that involves the elevated synthesis of a family of proteins commonly referred to as heat shock or stress-induced proteins. The HSP70 family participates in numerous protein biosynthetic reactions including the synthesis, translocation and folding of many cytoplasmic, organellar, membrane associated and secreted proteins. In this proposal we will investigate the properties and structure of the human HSP70 proteins. To accomplish this goal we will identify and characterize the peptide-binding domain of HSP70 by the construction of deletion and point mutants. In vitro studies will include peptide binding assays to examine and compare substrate specificities. Chimeric fusions between dnaK and human HSP70 and between the various members of the HSP70 family (p72/HSC70, GRP78/BiP and mitochondrial p75) will also be assayed in mammalian cells and in E. coli. The complementation assays in E. coli include lambda replication, growth of E. coli at elevated temperatures and autoregulation of the heat shock response. The function of the mutant HSP70 proteins, chimeric 7OkD stress proteins, HSP70-related proteins and bacterial dnaK will be examined in mammalian cells by redirecting their subcellular locale of these stress proteins and the analysis of subsequent in vivo interactions by immunofluorescence and immunoprecipitation assays. The collection of mutant heat shock proteins will be used to identify the amino acid sequence requirements for the cell cycle and stress-dependent reversible translocation of HSP70 between the cytosolic, nuclear and nucleolar compartments of the mammalian cell. Finally, we will clone the remaining (uncloned) HSP70-related genes in the human genome using sequence homology and monoclonal antibody reagents. This will allow us to establish whether distinct members of the HSP70 family differ in their biological and biochemical function.
| Freeman, B C; Myers, M P; Schumacher, R et al. (1995) Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO J 14:2281-92 |
| Bhattacharyya, T; Karnezis, A N; Murphy, S P et al. (1995) Cloning and subcellular localization of human mitochondrial hsp70. J Biol Chem 270:1705-10 |
| Murphy, S P; Gorzowski, J J; Sarge, K D et al. (1994) Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells. Mol Cell Biol 14:5309-17 |
| Sarge, K D; Park-Sarge, O K; Kirby, J D et al. (1994) Expression of heat shock factor 2 in mouse testis: potential role as a regulator of heat-shock protein gene expression during spermatogenesis. Biol Reprod 50:1334-43 |
| Morimoto, R I; Sarge, K D; Abravaya, K (1992) Transcriptional regulation of heat shock genes. A paradigm for inducible genomic responses. J Biol Chem 267:21987-90 |
