Identification and characterization of anovel factor that controls intracellular transport of peroxisomal proteins

通过对2,4 -二氯苯氧基丁酸的抗性，分离到拟南芥过氧化物酶体缺陷ped2突变体。在本项目中，通过定位克隆分离出了一个与该缺陷有关的基因，称为PED2基因，并通过互补分析证实了其身份。预测的蛋白产物的氨基酸序列与人类的Pex14p相似，Pex14p是过氧化物酶体蛋白进口机制的关键组成部分。因此我们决定将其命名为A/Pex14p。对ped2突变体的分析表明，A/Pex14p控制过氧化物酶体靶向信号（PTS） 1-和含有pts2的蛋白在细胞内转运到三种不同类型的过氧化物酶体，即乙氧基酶体、叶片过氧化物酶体和非特化过氧化物酶体。PED2基因的突变导致所有这些功能分化过氧化物酶体的酶减少。这些酶的减少引起多效性缺陷，如脂肪酸降解、光呼吸和过氧化物酶体的形态。这些数据表明，A/Pex14p通过决定过氧化物酶体蛋白的靶向性，在维持这三种植物过氧化物酶体的生理功能中具有共同的作用。有研究表明，拟南芥中Pex5p和Pex7p的同源基因也参与过氧化物酶体蛋白输入机制。免疫印迹分析显示，A/Pex14p和AfPex5p存在于拟南芥的大部分器官中，提示这些因子在植物过氧化物酶体的主要蛋白质输入途径中发挥作用。双杂化分析表明，4/Pex14p的两个氨基末端结构域（^<58>I-^<65>L和^<78>R-^<97>R）与4/Pex5p的氨基末端结构域（^<1>M-^<23>V）相互作用。AtPexl4p不与AtPex7p相互作用。相比之下，9个WXXXF/Y重复序列存在于atpex5p的^<231>K-^<450 >d中，与atpex7p的羧基端（^<266 >y -^<317 >s）具有结合活性。这些结果表明，含有PTS1 -和pts2的蛋白被含有4pex5p和AtPex7p的细胞质受体复合物捕获，然后通过与AtPex14p的结合被过氧化物酶体膜识别。少

Arabidopsis peroxisome-deficient ped2 mutant was isolated by its resistance to 2, 4-dichlorophenoxybutyric acid. In this project, a gene responsible for this deficiency, called the PED2 gene, was isolated by positional cloning and confirmed its identity by complementation analysis. The amino acid sequence of the predicted protein product is similar to that of human Pex14p, which is a key component of the peroxisomal protein import machinery. Therefore we decided to call it A/Pex14p. Analyzes of the ped2 mutant revealed that A/Pex14p controls intracellular transport of both peroxisome targeting signal (PTS) 1- and PTS2-containing proteins into three different types of peroxisomes, namely glyoxysomes, leaf peroxisomes and unspecialized peroxisomes. Mutation in the PED2 gene results in reduction of enzymes in all of these functionally differentiated peroxisomes. The reduction of these enzymes induces pleiotropic defects, such as fatty acid degradation, photorespiration and the morphology … More of peroxisomes. These data suggest that the A/Pex14p has a common role in maintaining physiological functions of each of these three kinds of plant peroxisomes by determining peroxisomal protein targeting. It had been suggested that Arabidopsis orthologues of Pex5p and Pex7p were also involved in peroxisomal protein import machinery. Immunoblot analysis showed that A/Pex14p and AfPex5p were present in most organs in Arabidopsis, suggesting that these factors play a role in the main protein import pathways for plant peroxisomes. Two-hybrid analysis showed that two amino-terminal domains (^<58>I-^<65>L and ^<78>R-^<97>R) of 4/Pex14p interacted with amino-terminus (^<1>M-^<23>V) of 4/Pex5p. AtPexl4p did not interact with AtPex7p. In contrast, the nine WXXXF/Y repeats existed in ^<231>K-^<450>D ofAtPex5p had binding activity with carboxyl terminus (^<266>Y-^<317>S) ofAtPex7p. These results indicated that PTS1 - and PTS2-containing proteins were captured by the cytosolic receptor complex containing 4tPex5p and AtPex7p, and then the receptor-cargo complex was recognized by the peroxisomal membrane by its binding with AtPex14p. Less