In normal cells, the tumor suppressor actions of p53 protein are mediated by specific DNA binding and protein-protein interactions within the nucleus. Mutant p53 protein, however, often assumes an aberrant conformation devoid of tumor suppressor activity and newly capable of cytoplasmic binding to HSP70 protein. We have identified components of the p53-HSP70 complex as well as the subcellular location of p53:HSP complexes in human and murine transformed cell lines containing aberrant p53 conformers. Anti-p53 immune complexes from cytoplasmic lysates contained not only HSC70 but also GRP75, GRP78 and HSP90 as proteins associated with p53 as identified by western blotting, immunoprecipitation and protein sequencing. Analysis of cytoplasmic HSP complexes has shown they exist as preformed multimers which may be important for recognition and binding.The presence of the mitochondrial and endoplasmic reticuluar chaperones, GRP75 and GRP78, in p53:HSP complexes suggested that p53 might be found in these cytoplasmic organelles which was confirmed in mitochondria by biochemical and immunoelectron microscopic evidence. These studies suggest that newly identified members of p53:HSP complexes represent components of a chaperone program which affects the subcellular distribution of p53 protein in these transformed lines.The research goal of this work is to understand the nature of the cellular response in HSP binding of aberrant p53 and to understand its role in the transformed phenotype. The underlying hypothesis is that aberrant p53 is recognized by preformed HSP complexes after exposure of cryptic binding sites. HSP binding to aberrant p53 is further hypothesized as a specific stress response to malfolded or misfolded p53 protein as an attempt to achieve correct folding or as part of degratory processing of mutant p53 proteins involved antigen presentation. An important portion of this work for protein identification has been through development of a preparative 2D PAGE system.