IMPORT, TARGETING, AND PROCESSING OF A PLANT POLYPHENOL OXIDASE

A tomato (Lycopersicon esculentum L.) gene encoding a precursor of pol yphenol oxidase (PPO) was transcribed and translated in vitro. The imp ort, targeting, and processing of the [S-35]methionine-labeled precurs or protein (pPPO) were studied in isolated chloroplasts. The protein w as routed to the thylakoid lumen in two steps. The 67-kD precursor was first imported into the stroma in an ATP-dependent step. It was proce ssed to a 62-kD intermediate by a stromal peptidase. Translocation int o the lumen was light dependent and involved processing of the 62-kD t o the 59-kD mature form. The mature polypeptide was soluble in the lum en and not bound to thylakoids. This two-step targeting pattern was ob served in plastids from a variety of plants including pea (Pisum sativ um L.), tomato, and maize (Zea mays L.). The ratio between the interme diate and mature forms observed depended on the plant species, leaf ag e, growth conditions, and illumination regime to which the plants had been subjected. Cu2+ was not required for pPPO import or processing. F urthermore, low concentrations of Cu2+ (1-5 mu M) markedly inhibited t he first import step. Tentoxin specifically inhibited pPPO import, lea ving the precursor bound to the envelope membrane. The two-step routin g of pPPO into chloroplasts, typical of thylakoid lumen proteins, is c onsistent with the two-domain structure of the transit peptide and app ears to be a feature of all plant PPO genes isolated so far. No eviden ce was found for unorthodox routing mechanisms, which have been sugges ted to be involved in the import of plant PPOs. The two-step routing m ay account for some of the multiplicity of PPO observed in vivo.