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

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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