Endopeptidases in the Stroma and Thylakoids of Pea Chloroplasts

Abstract

Three endopeptidases (EP2, EP3, and EP4) were identified after fractionation of pea (Pisum sativum, var Feltham First) chloroplast stromal extracts. All three were identified by their ability to cleave in vitro-synthesized preplastocyanin to lower molecular weight forms. EP2 is inhibited by phenylmethylsulfonylfluoride, and both EP2 and EP3 are inhibited by the heavy metal chelators 1,10-phenanthroline and EDTA. A further endopeptidase, EP5, was identified in Triton X-100 extracts of thylakoid membranes. Experiments involving contrifugation through a sucrose pad indicate that EP5 either has a high molecular weight or is associated with a thylakoid protein complex. EP5 is effectively inhibited by phenylmethylsulfonylfluoride, iodoacetate, and 1,10-phenanthroline, but not by EGTA. The implications of these results for the analysis of chloroplast protein maturation are discussed, and an improved protocol for the purification of the stromal processing peptidase is described which ensures the removal of EP2, the most active of the stromal peptidases analysed in this study.