The post-translational arginylation consists in the covalent union of an arginine residue to an acidic amino acid at the N-terminal position. Using mass spectrometry, we demonstrated in vitro the posttranslational incorporation of arginine into the ER calcium-binding chaperon calreticulin (CRT). To further study arginylated-CRT, we raised an antibody against a peptide corresponding to the N-terminal sequence of the arginylated-CRT, an arginine followed by the first 7 N-terminal amino acids of the mature rat CRT (RDPAIYFK). Western blot analysis from the soluble fraction of rat brain 14C-arginylated proteins in vitro showed that the anti arginylated-CRT antibody recognizes the 14C-arginylated-CRT, indicating that CRT incorporated 14C-arginine. We also show that arginylation of CRT is Ca2+ regulated: the level of arginylated-CRT in cell cultures increases upon the addition of Ca2+ chelator to the medium whereas no arginine is found in the presence of Ca2+. Besides, under stress conditions, arginylated-CRT is associated with stress granules. Finally, arginylated-CRT does not show the same sub-cellular distribution as CRT, which is located in the ER contrasting with the cytosolic localization of arginylated-CRT. Our results suggest that CRT arginylation occurs on a cytosolic pool of mature CRT that is most probably retro-translocated from the ER.
This work was supported in part by grants from the CONICET- FONCyt, SECYT UNC and from SECyT (
