A chloroplast processing enzyme functions as the general stromal processing peptidase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-06, Vol.95 (13), p.7463-7468
• Main Authors: Richter, S. (University of Chicago, IL.), Lamppa, G.K
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADN; Amino Acid Sequence; Amino acids; Antibodies; Apoproteins - metabolism; Arabidopsis; Arabidopsis Proteins; BINDING PROTEINS; Biological Sciences; Carrier proteins; CATION; CATIONES; CATIONS; Cell biology; CHLOROPHYLL A/B BINDING PROTEIN; CHLOROPHYLLE; CHLOROPHYLLS; Chloroplasts; Chloroplasts - enzymology; CINC; CLOROFILAS; COMPLEMENTARY DNA; DNA; ENDOPEPTIDASES; Enzymes; ENZYMIC ACTIVITY; ESCHERICHIA COLI; EXPRESION GENICA; EXPRESSION DES GENES; FERREDOXIN; GENBANK/AF008444; GENE EXPRESSION; GENE TRANSFER; LIGHT; LIGHT HARVESTING COMPLEXES; Light-Harvesting Protein Complexes; LUMIERE; LUZ; METAL; METALES; Metalloendopeptidases - metabolism; METALLOPROTEINE; METALLOPROTEINS; METALPROTEINAS; METALS; Metals - metabolism; MOLECULAR SEQUENCE DATA; NUCLEOTIDE SEQUENCE; Peas; PEPTIDASAS; PEPTIDASE; PEPTIDASES; Photosynthetic Reaction Center Complex Proteins - metabolism; Photosystem II Protein Complex; PISUM SATIVUM; Plant Proteins; PRECURSORS; Protein precursors; Protein Precursors - metabolism; Protein Processing, Post-Translational; PROTEINAS; PROTEINAS AGLUTINANTES; PROTEINAS RECOMBINANTES; PROTEINE; PROTEINE DE LIAISON; PROTEINE RECOMBINANTE; PROTEINS; PROTEOLISIS; PROTEOLYSE; PROTEOLYSIS; RECOMBINANT PROTEINS; Recombinant Proteins - metabolism; SECUENCIA NUCLEOTIDICA; Sequence Alignment; SEQUENCE NUCLEOTIDIQUE; SIGNAL PEPTIDE; TRANSFERENCIA DE GENES; TRANSFERT DE GENE; ZINC
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.13.7463

A highly specific stromal processing activity is thought to cleave a large 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 previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined 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 cleavage site demonstrating that both forms of the enzyme are sensitive to the 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 of other chloroplast proteins, and this activity depends on metal cations, such as zinc