A mechanism of release of calreticulin from cells during apoptosis

Abstract

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca2+ homeostasis. CRT also exerts extracellular functions, e.g., tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release remains unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here we show that under apoptotic stress conditions, the cytosolic CRT concentration increases and associates with phosphatidylserine (PS) in a Ca2+ dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT) which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH-groups by nitrogen species. During apoptosis, both CRT expression and the nitric oxide (NO) concentration increases. By using S-nitroso-L-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol (RSNO) dependent and caspase-independent manner. Furthermore, the CRT and PS relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS may explain how CRT acts as a bridging molecule during apoptotic cell clearance.