Subfractionation studies showed that cytochrome b₅ (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A–insensitive NADH–cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

We used a teleost fish, the mummichog (Fundulus heteroclitus), to conduct early life-stage toxicity testing for copper pyrithione (CuPT). Fertilized mummichog eggs were exposed to CuPT at various concentrations for 50 d under continuous flow-through conditions. Hatchability, survival, growth, and morphologic abnormalities were measured. Hatchability did not differ significantly between any experimental group and control groups. Survival and growth were significantly reduced at 50 d in the groups exposed to 2 or 4 μg/L CuPT. During the test, morphologic abnormalities, such as vertebral deformity and formation of inflammatory masses in the lateral muscles, occurred in fish exposed to CuPT. Light and electron microscopic studies indicated that muscle dysfunction played a role in the vertebral deformity and revealed that the inflammatory mass was composed mainly of macrophages and necrotic myocytes. We consider that macrophages infiltrated and phagocytized necrotic cells, thus forming the inflammatory mass. In addition, acetylcholinesterase activity was markedly decreased in the 2- and 4-μg/L exposure groups, suggesting the skeletal deformity was due to mechanisms similar to those proposed for organophosphorous pesticide exposure.