Abstract
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.
Highlights
One function of the ubiquitin (Ub)1-proteasome pathway is the selective degradation of abnormal proteins, as may arise by mutation or post-synthetic damage [1, 2]
To compare the conformational changes that occur upon Ca2ϩ binding in native CaM and in the in vitro aged species, we extended this approach, using two-dimensional gels that were run in the presence of EDTA in the first dimension and in the presence of either EDTA or Ca2ϩ in the second dimension
The low amounts of slowly migrating variants in native CaM are due to isomerization of aspartyl residues at positions outside the Ca2ϩ-binding regions [6, 17]
Summary
Purification of CaM—Recombinant chicken hemagglutinin-tagged CaM (HA-CaM) and metabolically labeled CaM ([35S]CaM) were purified from P4830 Escherichia coli cells that had been transformed with the pCaMpL plasmid containing the chicken CaM gene under the control of the phage left promoter. Degradation of CaM in HeLa Extract—Native or aged [35S]CaM (ϳ2 g) was added to a 50-l reaction containing 500 g of HeLa cell extract supplemented with an ATP-regenerating system (10 mM creatine phosphate, 0.2 mg/ml creatine kinase) and protease inhibitors (50 M E64, 1 mM phenylmethylsulfonyl fluoride, 50 M chymostatin) in Buffer H (Buffer H: 50 mM Na-HEPES, pH 7.5, 5 mM MgCl2, 1 mM ATP, 1 mM DTT) in the presence of 5 mM CaCl2 or EGTA. Ubiquitination Assay—Native or aged [35S]CaM (ϳ2 g in 50 mM Na-HEPES, pH 7.5, 1 mM EGTA) was added to a 20-l reaction containing 2 mM AMP-PNP, 30 M MG-132, 1.5 M ubiquitin aldehyde, 25 M ubiquitin, and 50 g of HeLa cell extract [37] in 20 mM Tris-Cl, pH 7.6, 20 mM KCl, 10 mM MgCl2, 1 mM DTT. The proteasomes were divided into two parts and used immediately for the degradation reactions: one part was supplemented with ATP (1 mM) and the other with buffer only
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