Ostersetzer, O., Department of Agricultural Botany, Hebrew University of Jerusalem, Rehovot, Israel
Tabak, S., Department of Agricultural Botany, Hebrew University of Jerusalem, Rehovot, Israel
Yarden, O., Department of Agricultural Botany, Hebrew University of Jerusalem, Rehovot, Israel
Shapira, R., Department of Agricultural Botany, Hebrew University of Jerusalem, Rehovot, Israel
Adam, Z., Department of Agricultural Botany, Hebrew University of Jerusalem, Rehovot, Israel, Department of Agricultural Botany, Hebrew University of Jerusalem, IL-76100 Rehovot, Israel

Despite numerous demonstrations of protein degradation in chloroplasts of higher plants, little is known about the identity of the proteases involved in these reactions. To identify chloroplast proteases by immunological means, we investigated two proteins: ClpP, a protein similar to the proteolytic subunit of the bacterial ATP-dependent Clp protease, for which a gene is found in the chloroplast genome [Maurizi, M.R., Clark, W.P., Kim, S.H. and Gottesman, S. (1990) J. Biol. Chem. 265, 12546-12552] and PrcA, a cyanobacterial Ca2+-stimulated protease [Maldener, I., Lockau, W., Cai, Y. and Wolk, P. (1991) Mol. and Gen. Genet. 225, 113-120]. We expressed the clpP gene from rice in Escherichia coli, purified its product, and generated antibodies against the product. Western blot analysis revealed the ClpP protein in different leaf extracts. Analysis of fractionated barley chloroplasts revealed that the protein was associated with the stromal fraction. The expression of ClpP is light independent and tissue specific, as it was found in green and etiolated barley leaves, but not in roots. A second protein, similar to the cyanobacterial protease PrcA, was also detected in chloroplasts. Antibody against this protease recognized proteins in various leaf extracts. When pea chloroplasts were fractionated, the antibody only recognized a stromal protein. The expression of this protein is regulated by light, as it was found in green leaves but not in etiolated leaves. The tissue specificity of PrcA was similar to that of ClpP in that it could not be detected in root extracts.