Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca2+

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca2+ 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 u p to 24 h in the ER (t(1/2) > 24 h). However, release of Ca2+ from the ER, initiated by the addition of inhibitors of the ER Ca2+/Mg2+ ATPase such as 2 mu M thapsigargin or 1 mu M ionomycin, initiated rapid loss of apoaequori n in the ER, but had no detectable effect on apoaequorin turnover in the cy tosol nor the nucleus. This loss of apoprotein was not the result of secret ion into the external fluid, and could not be inhibited by inhibitors of pr otein degradation by proteosomes. Proteolysis of apoaequorin in cell extrac ts (t(1/2) < 20 min) was completely inhibited in the presence of 1 mM Ca2+, and this effect was independent of the ER retention signal KDEL at the C-t erminus. Proteolysis was unaffected by the presence of selected serine prot ease inhibitors, or 10 mu M Zn2+, a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by l oss of ER Ca2+. Several Ca2+- binding proteins exist in the ER, acting as t he Ca2+ store and chaperones. Our results have important implications both for the role of ER Ca2+ in cell activation and stress and when using aequor in for monitoring free ER Ca2+ over long time periods. (C) 2000 Academic Pr ess.