TARGETING AEQUORIN AND GREEN FLUORESCENT PROTEIN TO INTRACELLULAR ORGANELLES

Two proteins of Aequorea victoria were molecularly engineered and prod uced in mammalian cells, in order to serve as specific reporters of su bcellular microenvironments. Aequorin (AEQ), a Ca2+-sensitive photopro tein, was successfully targeted to three intracellular locations: cyto sol, nucleus and mitochondria. The recombinant apoprotein, reconstitut ed into active AEQ by the addition of the prosthetic group to the cult ure medium, allows the direct measurement of [Ca2+] within those compa rtments, thus directly addressing questions of large biological intere st. The same approach was utilized for the green fluorescent protein ( GFP) for specific labelling, in vivo, of the various subcellular struc tures, GFP was targeted to mitochondria: the recombinant protein, stro ngly fluorescent in a highly reducing environment, provides a powerful tool for visualizing these organelles in living cells, and may repres ent the prototype of a new family of intracellularly targeted fluoresc ent probes.