Differences in the proteasomal degradation between calreticulin, arginylated calreticulin and non-arginylatable calreticulin

GOITEA VICTOR E.; HALLAK MARTA E.

Capital Federal

Congreso; Ubiquitin and ubiquitin-like proteins: At the crossroads from chromatin to protein; 2014

EMBO

Differences in the proteasomal degradation between calreticulin, arginylated calreticulin and non-arginylatable calreticulin Goitea V.E. and Hallak M.E. Centro de Investigaciones en Química Biológica de Córdoba CIQUIBIC-CONICET, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Córdoba - Argentina E-mail: vgoitea@fcq.unc.edu.ar Arginylation is a posttranslational modification that occurs at the N-terminal position of proteins, catalyzed by Ate1. The addition of Arg was involved in the N-end rule, as a primary destabilizing residue. Calreticulin (CRT) is retrotranslocated from the endoplasmic reticulum to the cytoplasm, where it is arginylated (R-CRT). We have previously demonstrated that R-CRT associates with stress granules (SGs) under stress conditions. Interestingly, the SGs formation under proteasome inhibition has been proved efficient for the treatment of myelomas and other hematological tumors. Here, we evaluate if R-CRT is ubiquitinated and degraded by the proteasome. We developed stable cell lines expressing CRT (arginylatable), R-CRT (arginylated), or E1V-CRT (non-arginylatable) in the cytoplasm. These cells in the presence of the proteasome inhibitor MG132 show an increased levels of EGFP-tagged proteins compared with control cells. Moreover, we determine the degradation rates in the presence of cycloheximide to inhibit the protein synthesis. Our results indicate that the half-life of the three isoforms are different, indicating a different susceptibility to proteasomal degradation. Furthermore, we performed an in vivo ubiquitination assay to determine the ubiquitination status R-CRT and E1V-CRT. Our results indicate that arginylation of CRT is necessary for its ubiquitination and that CRT could be degraded by the proteasome in an ubiquitin independent manner. Supported by SECyT-UNC, CONICET, ANPCyT