CARBOXYL METHYLATION OF DEAMIDATED CALMODULIN INCREASES ITS STABILITYIN XENOPUS OOCYTE CYTOPLASM - IMPLICATIONS FOR PROTEIN REPAIR

The widely distributed protein-L-isoaspartate(D-aspartate) O-methyltra nsferase (PIMT; EC 2.1.1.77) is postulated to play a role in the repai r or metabolism of damaged cellular proteins containing L-isoaspartyl residues derived primarily from the spontaneous deamidation of protein asparaginyl residues. To evaluate the functional consequence of PIMT- catalyzed methylation on the stability of isoaspartyl-containing prote ins in cells, Xenopus laevis oocytes were microinjected with both deam idated and nondeamidated forms of recombinant chicken calmodulin (CaM) containing a hemagglutinin (HA) epitope at its N terminus. Processing of HA-CaM was monitored by electrophoretic analysis and Western blott ing of oocyte extracts. The experiments indicate that deamidated HA-Ca M is degraded after microinjection, while nondeamidated HA-CaM is stab le. Kinetic analysis is consistent with the entry of microinjected HA- CaM into two intracellular pools with distinct hydrolytic stabilities. The larger, more stable pool may consist of HA CaM bound to the heter ogeneous pool of oocyte CaM binding proteins detected by an overlay pr ocedure. Enzymatic methylation of deamidated HA-CaM with purified PIMT prior to injection results in its stabilization. Conversely, inhibiti on of endogenous oocyte PIMT with sinefungin, a nonhydrolyzable analog of S-adenosylhomocysteine, increases the rate of deamidated HA-CaM de gradation. These results are consistent with a role for PIMT-catalyzed methylation in the repair of damaged cellular proteins.