CONICET | Buscador de Institutos y Recursos Humanos

Intracellular localization of calreticulin studied using a biological approach Máximo López Medus, Ana Villamil Giraldo, Rodrigo Sebastian Pagano, Carlos Labriola, Lucas Landolfo, Armando Parodi and Julio Javier Caramelo Calreticulin (CRT) is an abundant protein resident of endoplasmic reticulum (ER) that works as a lectin-chaperone and is one of the main intracellular calcium buffers. Although CRT displays an export peptide and an ER retrieval signal, several reports have found it in the cytosol. By using a biochemical approach we previously reported that the presence of CRT in the cytosolic fraction is regulated by ER calcium levels. In order to gain further insight into the mechanism of translocation, we used CRT GAL4-P53, a fusion protein that was expressed in mamallian cells (COS-7). GAL4 is a DNA binding domain of the yeast protein while P53 is a classical activator of POL II. We cotransfected these cells with GAL-P53-dependent luciferase reporter plasmid, which has the firefly luciferase gene downstream a promoter and UAS region. When the GAL4 DBD present in the fusion protein binds this region the luciferase gene switches on. Using this system, we were able to indirectly measure the amount of CRT GAL-P53 present in the cytoplasm by measuring the level of luciferase activation. Under conditions in which the ER is depleted of Ca2+, i.e. after a 30 min treatment with thapsigargin which inhibits the ER Ca2+ pump, the signal was increased by 10-20%. This alternative approach provided further evidence supporting the modulation of cytoplasmic CRT levels by ER Ca2+ concentration.