ATP-dependent association between subunits of Clp protease in pea chloroplasts

The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of th e proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both s ubunits are found in the stroma, the interaction between the two is dynamic . When immunoprecipitation with antibodies against ClpC was performed on st roma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-T-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma w as fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma deple ted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrat ed separately, whereas in the presence of ATP-gamma -S both subunits co-mig rated. Similar results were observed in size-exclusion chromatography. To f urther characterize the precipitated enzyme, its proteolytic activity was a ssayed by testing its ability to degrade fl-casein. No degradation was obse rved in the absence of ATP, and degradation was inhibited in the presence o f phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested us ing chimeric OE33 as a model substrate. This protein was also degraded in a n ATP-dependent manner, supporting the suggested role of Cip protease as a major housekeeping protease in the stroma.