DEGRADATION OF MISTARGETED OEE33 IN THE CHLOROPLAST STROMA

OEE33, a component of the oxygen-evolving enzyme in chloroplasts, norm ally resides in the thylakoid lumen. In an attempt to study the fate o f mistargeted proteins in chloroplasts, we substituted the bipartite t ransit peptide of OEE33 with that of CAB7, an integral thylakoid-membr ane protein. As a result, when imported into isolated chloroplasts, th e chimeric protein was targeted to the stroma instead of the thylakoid lumen. Whereas the wild-type OEE33 was totally stable for at least 2 h, the chimeric protein was rapidly degraded, with a half-life of 60 m in. Degradation of the chimeric protein was stimulated by ATP suppleme ntation. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteoly tic activity was found to be associated mainly with the stromal fracti on. This activity was very effectively inhibited by all tested inhibit ors of serine proteases. Western blot analysis demonstrated that the s tromal fraction active in degrading the chimeric OEE33 contains ClpC a nd ClpP, homologues of the regulatory and proteolytic subunits, respec tively, of the bacterial, ATP-dependent, serine-type Clp protease.