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-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.
Tarcsa
E
E
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Szymanska
G
G
Lecker
S
S
O'Connor
C M
CM
Goldberg
A L
AL
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002645:Chickens; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections; D018482:Muscle, Skeletal / Q000201:enzymology; D009865:Oocytes / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D014452:Ubiquitins / Q000378:metabolism; D014981:Xenopus
D002147:Calmodulin; D014452:Ubiquitins; C067713:lactacystin; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D002118:Calcium; D000111:Acetylcysteine