Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H 2 O 2 and b-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein-protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants.

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SUMMARYPEX7 is a soluble import receptor that recognizes peroxisomal targeting signal type 2 (PTS2)-containing proteins. In the present study, using a green fluorescent protein (GFP) fusion protein of PEX7 (GFP-PEX7), we analyzed the molecular function and subcellular localization of PEX7 in Arabidopsis thaliana. The overexpression of GFP-PEX7 resulted in defective glyoxysomal fatty acid b-oxidation, but had no significant effect on leaf peroxisomal function. Analysis of the subcellular localization of GFP-PEX7 in transgenic Arabidopsis showed that GFP-PEX7 localizes primarily to the peroxisome. Transient expression of a C-or N-terminal fusion protein of PEX7 and yellow fluorescent protein (YFP) (PEX7-YFP and YFP-PEX7, respectively) in leek epidermal cells, using the particle bombardment technique, confirmed that fluorescent protein-tagged PEX7 localizes to peroxisomes in Arabidopsis. Immunoblot analysis revealed that GFP-PEX7 accumulates primarily in peroxisomal membrane fractions, whereas endogenous PEX7 was distributed evenly in cytosolic and peroxisomal membrane fractions, which indicated that both endogenous PEX7 and GFP-PEX7 are properly targeted to peroxisomal membranes. The results of bimolecular fluorescence complementation (BiFC) and yeast two-hybrid analyses showed that PEX7 binds directly to PTS2-containing proteins and PEX12 in the peroxisomal membrane. We used red fluorescent protein (tdTomato) fusion protein of PEX7 (tdTomato-PEX7) in several Arabidopsis pex mutants to identify proteins required for the targeting of PEX7 to peroxisomes in planta. The results demonstrated that pex14, pex13 and pex12 mutations disrupt the proper targeting of PEX7 to peroxisomes. Overall, our results suggest that the targeting of PEX7 to peroxisomes requires four proteins: a PTS2-containing protein, PEX14, PEX13 and PEX12.