ROLE OF Ca2+ IN THE AGGREGATION OF ARGINYLATED CALRETICULIN WITHIN THE STRESS GRANULES

CARPIO, MARCOS A; LOPEZ SAMBROOKS, CECÍLIA; HALLAK, MARTA

Viña del Mar, Chile

Workshop; 8TH INTERNATIONAL CALRETICULIN WORKSHOP; 2009

We have previously shown that arginine can be posttranslationally incorporated into the NH2-terminus of soluble aceptor proteins by arginyl-tRNA protein transferase (Ate1). Using mass spectrometry, we have also demonstrated in vitro the posttranslational incorporation of arginine into the calcium-binding chaperone calreticulin (CRT). To further characterize this arginylated isospecies we performed in vitro studies in cells lacking of Ate1 (Ate1-/-) (gift of Dr. Kashina). In these cells CRT was not arginylated as in Ate1+/+, an indication that this enzyme arginylates CRT.By immunocytochemistry was shown that the arginylated isoform of calreticulin (R-CRT) showed a cytosolic localization that is different from the CRT present in the endoplasmic reticulum. As a control, the analysis of CRT-/- cells (kindly provided by Dr. Michallak) showed that both, CRT and R-CRT were not detected by anti-CRT and anti-R-CRT antibodies, respectively. Therefore CRT arginylation occurs on a cytosolic pool of mature CRT that is most probably retro-translocated from the ER.We also showed that arginylation of CRT is regulated in vitro by Ca2+: the addition of Ca2+ chelator to the medium increases the level of arginylated-CRT. Furthermore, we found that the cytosolic calcium decreases in NIH3T3 cells exposed to Tg-EGTA, heat shock or oxidative stress and in correlation R-CRT is increased. These environmental stress conditions that activated the unfolded protein response and the appearence of the stress granules (SGs, the sites of acumulation of stalled translation initiation complexes) also resulted in the aggregation of R-CRT and its association with SGs. We concluded that posttranslational arginylation of CRT can regulate its intracellular localization and perhaps its cytosolic function no yet determined.Supported by SECyT-UNC, CONICET, ANPCyT-PICT