Targeting aequorin and green fluorescent protein to intracellular organelles.
Two proteins of Aequorea victoria were molecularly engineered and produced in mammalian cells, in order to serve as specific reporters of subcellular microenvironments. Aequorin (AEQ), a Ca(2+)-sensitive photoprotein, was successfully targeted to three intracellular locations: cytosol, nucleus and mitochondria. The recombinant apoprotein, reconstituted into active AEQ by the addition of the prosthetic group to the culture medium, allows the direct measurement of [Ca2+] within those compartments, thus directly addressing questions of large biological interest. The same approach was utilized for the green fluorescent protein (GFP) for specific labelling, in vivo, of the various subcellular structures. GFP was targeted to mitochondria: the recombinant protein, strongly fluorescent in a highly reducing environment, provides a powerful tool for visualizing these organelles in living cells, and may represent the prototype of a new family of intracellularly targeted fluorescent probes.
De Giorgi
F
F
Department of Biomedical Sciences, University of Padova, Italy.
Brini
M
M
Bastianutto
C
C
Marsault
R
R
Montero
M
M
Pizzo
P
P
Rossi
R
R
Rizzuto
R
R
D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D015388:Organelles / Q000378:metabolism*; D037401:Scyphozoa; D013347:Subcellular Fractions
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000331:Aequorin; D002118:Calcium