Ultrastructural localization of Hsp-72 examined with a new polyclonal antibody raised against the truncated variable domain of the heat shock protein

In spite of the intensive search for the determination of the continuously widening physiological and pathological roles of different stress proteins, their ultrastructural localization at the electron microscopic (EM) level has hardly been examined. As it becomes increasingly evident that the funct ion and physiological effectiveness of stress proteins are highly dependent on their spatial location and their associations with diverse regulator pr oteins, the demand for morphological studies which can identity their detai led distribution within the cells is evident. The reason for the practical lack of studies carried out at the EM level, lies in the shortage of reagen ts with suitable specificity and avidity necessary for this type of examina tion. To create such a reagent, a polyclonal antibody was raised using a re combinant truncated form of the inducible Hsp-72 protein. The antibody was extensively characterized, using different immunochemical methods to determ ine and verify its specificity, and then it was tried in ultrastructural ex aminations. Using the new antibody, it was possible to analyze the intracel lular distribution of Hsp-72 with the immunogold technique. The localizatio n of Hsp-72 was demonstrated directly at the ultrastructural level in the c ytoplasm (especially at the cisterns of the RER), in the nucleus (mainly ar ound the heterochromatic regions) and at both sides of the nuclear envelope close to the membrane pores. Apart from these localizations, Hsp-72 was fo und in several membrane bordered intracellular structures, which mainly bel ong to the endosomal-lysosomal system. We provide the first morphological v erification of the appearance of Hsp-72 on the surface of the cells. Also n ovel is the indication, that the stress protein may recycle from the cell s urface using a common route which includes coated pits and the endosomal sy stem.