The heat shock or cellular stress response is a highly conserved cellular response to a variety of potentially lethal physical, chemical, and biological agents. The broad longterm objectives of this research program are to determine the functions of members of stress protein families that have roles in the normal cellular physiology of vertebrates. The project focuses on the 73 kilodalton mammalian heat shock cognate protein, hsc73, that can disassemble clathrin cages in vitro and that promotes tranlocation of several proteins across endoplasmic reticulum and mitochondrial membranes. Models for the mechnism by which hsp70 family members may repair damaged structures such as nucleoli, solubilize aggregates of denatured proteins, or promote translocation of other mproteins across membranes frequently invoke conformational changes. However, no direct evidence of protein conformational changes has been obtained, nor has the protein binding site(s) on any hsp70 family member been characterized, nor are the features of the interactive proteins involved in bninding to hsp70 family members known. The experiments proposed will directly address the first two gaps in our understanding of this protein family and will forge the way to filling the third gap. Two novel reagents that have not been applied previously to studies of this protein family are featured in this application, a small well-characterized protein apocytochrome c that binds hsc73, and a vector construct capable of expressing functional rat hsc73 in E. coli. Biophysical studies of the hsp70 proteins are almost nonexistent, and so another goal is to obtain additional spectra using circular dichroism as part of a search for evidence of conformational changes and provide basic physical data on hsc73 such as exteinction coefficient and per cent helical content. Inducible eucaryotic expression vectors have been constructed to produce hsc73 antisense transcripts and to overproduce hsc73. Mammalian trnsformants have been developed and studies are planned to identify and anlyze phenotypes associated with cellular deficiencies in and overproduction of hsc73. This is a multidisciplinary approach to the question of the function of heat shock response proteins. The heat shock response, or stress response, is a ubiquitous response of eukaryotic cells to a broad variety of stresses. Although there has been a great deal of knowledge developed in terms of cataloguing the proteins which are either induced or up-regulated in response to stress, the functions of these proteins remain unknown.
