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

Jeffery, J., Kendall, J. M. and Campbell, Anthony Keith 2000. Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca2+. Biochemical and Biophysical Research Communications 268 (3) , pp. 711-715. 10.1006/bbrc.2000.2194

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Abstract

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmicreticulum of HeLa cells in order to determine the effect of Ca2+ release from the ER on proteindegradation. 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 (t1/2 > 24 h). However, release of Ca2+ from the ER, initiated by the addition of inhibitors of the ER Ca2+/Mg2+ ATPase such as 2 μM thapsigargin or 1 μM 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 proteindegradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t1/2 < 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-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 μM Zn2+, a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ERproteins activated by loss of ER Ca2+. Several Ca2+-binding proteins exist in the ER, acting as the Ca2+ store and chaperones. Our results have important implications both for the role of ER Ca2+ in cell activation and stress and when using aequorin for monitoring free ER Ca2+ over long time periods.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Pharmacy
Subjects: Q Science > QD Chemistry
R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords: calcium; aequorin; endoplasmicreticulum; protein turnover
Publisher: Elsevier
ISSN: 0006-291X
Last Modified: 05 Jun 2017 02:58
URI: http://orca-mwe.cf.ac.uk/id/eprint/22525

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