IMMUNOLOGICAL DETECTION OF PROTEINS SIMILAR TO BACTERIAL PROTEASES INHIGHER-PLANT CHLOROPLASTS

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 imm unological means, we investigated two proteins: ClpP, a protein simila r to the proteolytic subunit of the bacterial ATP-dependent Clp protea se, for which a gene is found in the chloroplast genome [Maurizi, M. R ., Clark, W.P., Kim, S. H. & Gottesman, S. (1990) J. Biol. Chem. 265, 12546-12552] and PrcA, a cyanobacterial Ca2+-stimulated protease [Mald ener, I., Lockau, W., Cai, Y. & Wolk, P. (1991) Mel. & Cen. Genet. 225 , 113-120]. We expressed the clpP gene from rice in Escherichia coli, purified its product, and generated antibodies against the product. We stern blot analysis revealed the ClpP protein in different leaf extrac ts. Analysis of fractionated barley chloroplasts revealed that the pro tein 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 e xtracts. When pea chloroplasts were fractionated, the antibody only re cognized a stromal protein. The expression of this protein is regulate d by light, as it was found in green leaves but not in etiolated leave s. The tissue specificity of PrcA was similar to that of ClpP in that it could not be detected in root extracts.