Sommer A, Ne'eman E, Steffens J C, Mayer A M, Harel E
Botany Department, Hebrew University, Jerusalem, Israel.
Plant Physiol. 1994 Aug;105(4):1301-11. doi: 10.1104/pp.105.4.1301.
A tomato (Lycopersicon esculentum L.) gene encoding a precursor of polyphenol oxidase (PPO) was transcribed and translated in vitro. The import, targeting, and processing of the [35S]methionine-labeled precursor protein (pPPO) were studied in isolated chloroplasts. The protein was 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 processed to a 62-kD intermediate by a stromal peptidase. Translocation into the lumen was light dependent and involved processing of the 62-kD to the 59-kD mature form. The mature polypeptide was soluble in the lumen and not bound to thylakoids. This two-step targeting pattern was observed in plastids from a variety of plants including pea (Pisum sativum L.), tomato, and maize (Zea mays L.). The ratio between the intermediate and mature forms observed depended on the plant species, leaf age, growth conditions, and illumination regime to which the plants had been subjected. Cu2+ was not required for pPPO import or processing. Furthermore, low concentrations of Cu2+ (1-5 microM) markedly inhibited the first import step. Tentoxin specifically inhibited pPPO import, leaving the precursor bound to the envelope membrane. The two-step routing of pPPO into chloroplasts, typical of thylakoid lumen proteins, is consistent with the two-domain structure of the transit peptide and appears to be a feature of all plant PPO genes isolated so far. No evidence was found for unorthodox routing mechanisms, which have been suggested to be involved in the import of plant PPOs. The two-step routing may account for some of the multiplicity of PPO observed in vivo.
一个编码多酚氧化酶（PPO）前体的番茄（Lycopersicon esculentum L.）基因在体外进行了转录和翻译。对[35S]甲硫氨酸标记的前体蛋白（pPPO）在分离的叶绿体中的导入、靶向和加工过程进行了研究。该蛋白分两步被转运到类囊体腔中。67-kD的前体首先通过一个依赖ATP的步骤被导入基质。它被一种基质肽酶加工成62-kD的中间体。向腔内的转运依赖于光，并涉及将62-kD加工成59-kD的成熟形式。成熟的多肽可溶于腔内，不与类囊体结合。在包括豌豆（Pisum sativum L.）、番茄和玉米（Zea mays L.）在内的多种植物的质体中都观察到了这种两步靶向模式。观察到的中间体和成熟形式之间的比例取决于植物种类、叶龄、生长条件以及植物所经历的光照条件。pPPO的导入或加工不需要Cu2+。此外，低浓度的Cu2+（1-5 microM）显著抑制了第一步导入。抗霉素A特异性地抑制pPPO的导入，使前体与包膜膜结合。pPPO进入叶绿体的两步转运，是类囊体腔蛋白的典型特征，与转运肽的双结构域结构一致，似乎是迄今为止分离到的所有植物PPO基因的一个特征。没有发现证据表明存在非传统的转运机制，而此前有人认为这种机制参与了植物PPO的导入。这种两步转运可能解释了体内观察到的PPO多样性的部分原因。
