Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca(2+).
Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca(2+) release from the ER on protein degradation. In resting cells apoaequorin had a rapid half-life (ca. 20-30 min) in the cytosol or nucleus, but was relatively stable for up to 24 h in the ER (t(1/2) > 24 h). However, release of Ca(2+) from the ER, initiated by the addition of inhibitors of the ER Ca(2+)/Mg(2+) ATPase such as 2 microM thapsigargin or 1 microM ionomycin, initiated rapid loss of apoaequorin in the ER, but had no detectable effect on apoaequorin turnover in the cytosol nor the nucleus. This loss of apoprotein was not the result of secretion into the external fluid, and could not be inhibited by inhibitors of protein degradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t(1/2) < 20 min) was completely inhibited in the presence of 1 mM Ca(2+), and this effect was independent of the ER retention signal KDEL at the C-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 microM Zn(2+), a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by loss of ER Ca(2+). Several Ca(2+)-binding proteins exist in the ER, acting as the Ca(2+) store and chaperones. Our results have important implications both for the role of ER Ca(2+) in cell activation and stress and when using aequorin for monitoring free ER Ca(2+) over long time periods.
Jeffery
J
J
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, United Kingdom.
Kendall
J M
JM
Campbell
A K
AK
D000331:Aequorin / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D001059:Apoproteins; D002135:Calcium-Binding Proteins; D011506:Proteins; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin; D010450:Endopeptidases; D002118:Calcium