A CHLOROPLAST PROCESSING ENZYME FUNCTIONS AS THE GENERAL STROMAL PROCESSING PEPTIDASE

A highly specific stromal processing activity is thought to cleave a l arge diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established, We have previ ously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll alb binding protein of photosystem II (LHCPII). Here we report the overexpression of acti ve CPE in Escherichia coil, Examination of the recombinant enzyme in v itro revealed that it cleaves not only preLHCPII, but also the precurs ors for an array of proteins essential for different reactions and des tined for different compartments of the organelle, CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleava ge site demonstrating that both forms of the enzyme are sensitive to t he same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase, Significantly, recombinant CPE cleaves in the absence o f other chloroplast proteins, and this activity depends on metal catio ns, such as zinc.