Targeting aequorin to the endoplasmic reticulum of living cells.
The photoprotein aequorin has been engineered with an ER targeting sequence at the N-terminus, with and without KDEL at the C-terminus, so that it locates in the ER-secretory pathway. For the first time the free Ca2+ has been quantified inside the ER and shown to be 5-20 times that in the cytosol. In COS cells free Ca2+ in the ER ranged from 1-5mM at 37 degrees C, decreasing 2-5-fold within 1 min of exposure to the Ca2+ ionophore ionomycin in the absence of external Ca2+.
Kendall
J M
JM
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Dormer
R L
RL
Campbell
A K
AK
D000331:Aequorin / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics; D037282:Calreticulin; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D037401:Scyphozoa / Q000235:genetics; D014162:Transfection; D001618:beta-Lactamases / Q000235:genetics
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000331:Aequorin; D015759:Ionomycin; D004247:DNA; D001618:beta-Lactamases; D002118:Calcium