A mechanism of release of calreticulin from cells during apoptosis
Journal of Molecular Biology
This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jmb.2010.06.064.
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.
We thank Dagmara Szestakowska for doing the pilot work for this study. This work was supported by Arthritis Research UK grants E0521, E0543, 17231, 17966 and 16537. PW, PE are grateful for support in the form of a European Union FP7 Marie Curie ITN grant (no. 215009). P.J.Y. and R.M. were supported by the Vandervell Foundation. P.J.Y. and D.J.S. are grateful for support by grants from Fight SMA and the SMA Trust. P.J.Y. and P.E. are thankful for a grant from the Northcott Devon Medical Foundation which funded the cloning work in this study. S.J. is supported by UK MRC grant (G0400389). J.T. thanks PMS for a PhD studentship. We thank Drs Tyurina and Stoyanovski of the University of Pittsburgh for advice on SNCEE synthesis and Dr Giles Cory for critical reading of the manuscript.
Vol. 401, Iss. 5, 3 September 2010, pp. 799–812