Induction and subcellular localization of two major stress proteins in response to copper in the fathead minnow Pimephales promelas

Abstract

In the present study we characterize the stress response induced by copper in the fathead minnow, Pimephales promelas. The fathead minnow epithelial cell line ATCC CCL 42 was used to examine the induced synthesis and subcellular localization of the two major stress proteins, stress70 and cpn60. Western blot analysis demonstrated increased stress70 in cells exposed to 400 and 500 μM Cu. Two-dimensional analysis revealed three isoforms of stress70, one of 70 kDa and two of 72 kDa, at the highest Cu concentration. Chaperonin60 abundance did not change over the same range of Cu concentrations. Indirect immunofluorescence microscopy revealed that stress70 localized in the cytoplasm, particularly in the paranuclear region. Chaperonin60 was localized in mitochondria. Further, when we examined the stress response elicited by Cu in fathead minnow larvae in vivo, we found that Cu induced the stress response at nominal Cu concentrations that were more than an order of magnitude lower that in the cell culture. This disparity between the concentration of Cu, which induced the stress response in cells in culture and in vivo, may be the result of differences in Cu complexation that alter its availability, uptake and toxicity.