Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without ubiquitination

The ubiquitin-proteasome pathway is believed to selectively degrade post-sy nthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regu lation and because it acquires isoaspartyl residues in its Ca2+-binding reg ions both in vivo and after in vitro "aging" (incubation for 2 weeks withou t Ca2+). When microinjected into Xenopus oocytes, in vitro aged CaM was deg raded much faster than native CaM by a proteasome-dependent process. Simila rly, in HeLa cell extracts aged CaM was degraded at a higher rate, even tho ugh it was not conjugated to ubiquitin more rapidly than the native species . Ca2+ stimulated the ubiquitination of both species, but inhibited their d egradation. Thus, for CaM, ubiquitination and proteolysis appear to be diss ociated. Accordingly, purified muscle 26 S proteasomes could degrade aged C aM and native Ca2+-free (apo) CaM without ubiquitination. Addition of Ca2dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca2+ binding, native CaM assumes a non-degradable conformation, which most of the age-damaged sp ecies cannot assume. Thus, flexible conformations, as may arise from age-in duced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.