Purification and properties of a novel chloroplast stromal peptidase. Processing of polyphenol oxidase and other imported precursors.
Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the DeltapH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.
Department of Plant Sciences, the Hebrew University, Jerusalem 91904, Israel.
S
S
Koussevitzky
E
E
Ne'eman
A
A
Sommer
J C
JC
Steffens
E
E
Harel
D000595:Amino Acid Sequence; D001692:Biological Transport; D004156:Catechol Oxidase / Q000378:metabolism*; D002451:Cell Compartmentation; D002736:Chloroplasts / Q000201:enzymology*; D018551:Solanum lycopersicum; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D018532:Pisum sativum; D010940:Plant Proteins; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D013379:Substrate Specificity
D010940:Plant Proteins; D011498:Protein Precursors; D004156:Catechol Oxidase; D008666:Metalloendopeptidases; C071986:stromal processing peptidase