CONICET | Buscador de Institutos y Recursos Humanos

Posttranslational arginylation of proteins consists in the covalent union of arginine in an acidic amino acid at the N-terminus position. We demonstrated that both in vitro and in vivo assays calreticulin (CRT) is modify by arginylation. Arginylated calreticulin (R-CRT) showed a different subcellular localization that the one described for CRT within the ER. In this work we show that transfected CRT-/- cells with full length CRT containing the ER signal peptide, leads to the appearance of a cytoplasmic pool of R-CRT. Since the site for arginylation of CRT arises from the cleavage of the signal peptide in the ER, the increased substrate in the cytoplasm must be a consequence of CRT retrotranslocation from the ER. We also confirmed that once CRT is in the cytoplasm it is posttranslationally argininated by ATE1. In previous experiments in vitro and in cell culture, was shown that CRT arginylation is inhibited by Ca2+. It has been demonstrated that in the ER CRT adopts a compact conformation and has a protease resistant core identified as the N-terminus region (Corbett, 2006). In this work we show that R-CRT is resistant to trypsin digestion compared to ER-CRT, even in the absence of Ca2+. In conclusion, we propose that CRT arginylation could be affecting the trypsin resistant digestion due to a conformational change. Supported by SECyT-UNC, CONICET, ANPCyT-PICT.