Nucleoplasmin-targeted aequorin provides evidence for a nuclear calcium barrier.
The Ca(2+)-activated photoprotein aequorin has been targeted to the nucleus of live cells by engineering nucleoplasmin, a nuclear structural protein from Xenopus laevis, onto the amino terminus. Successful targeting of the apoprotein was demonstrated by immunolocalization and selective permeabilization of the plasma membrane. Functional protein was reconstituted in live cells by incubation with coelenterazine. The effect of elevating cytosolic Ca2+ on nuclear Ca2+ was investigated in populations of live COS7 cells expressing either cytosolic aequorin or nuclear aequorin. Incubation of cells with ionomycin, in the absence of external Ca2+, released Ca2+ from internal stores causing an increase in chemiluminescent light emission from cytosolic aequorin but not nuclear aequorin. Nonagonist-dependent movement of Ca2+ across the plasma membrane, induced by the membrane attack complex of complement, causes a large increase in cytosolic Ca2+ which triggered 95% of cytosolic aequorin but only 50-60% of nuclear aequorin. These results provide clear evidence for a nucleocytoplasmic barrier to Ca2+.
Badminton
M N
MN
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.
Kendall
J M
JM
Sala-Newby
G
G
Campbell
A K
AK
D000331:Aequorin / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D015938:Complement Membrane Attack Complex; D003600:Cytosol / Q000378:metabolism; D007093:Imidazoles; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D056489:Nucleoplasmins; D010750:Phosphoproteins; D011719:Pyrazines; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism
D000957:Antigens, Viral, Tumor; D001059:Apoproteins; D015938:Complement Membrane Attack Complex; D007093:Imidazoles; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D011719:Pyrazines; D011993:Recombinant Fusion Proteins; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium