Abstract
Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm.
Highlights
Calreticulin (CRT) retrotranslocated from the endoplasmic reticulum to the cytoplasm is post-translationally arginylated
In CRT-EGFP-expressing cells treated with MG132, protein accumulation was detected with anti-GFP at essentially the same time that Results: Arginylated CRT (R-CRT) accumulation was detected by Western blotting with anti-RCRT Ab (Fig. 4F)
These findings indicate that proteasome inhibition results in accumulation of R-CRTEGFP, CRT-EGFP, and E1V-CRT-EGFP and suggested that (i) CRT arginylation is not required for its degradation, and (ii) R-CRT-EGFP and E1V-CRT-EGFP are less susceptible to proteasome degradation than is CRT-EGFP
Summary
Calreticulin (CRT) retrotranslocated from the endoplasmic reticulum to the cytoplasm is post-translationally arginylated. Significance: Arginylation is not required for proteasomal degradation of CRT, R-CRT displays ubiquitin modification. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). To assess the effect of CRT arginylation on its stability, we studied the degradation of cytoplasmic CRT (cyt-CRT) and R-CRT in fibroblasts and CHO cells, including the roles of proteasomes, Ub modification, and dimer formation in this process. Our finding that arginylation stabilizes CRT is in contrast to the traditional view of arginylation as a destabilizing factor
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