Ubiquitin-conjugating activity by PEX4 is required for efficient protein transport to peroxisomes in Arabidopsis thaliana.

Protein transport to peroxisomes requires various proteins, such as receptors in the cytosol and components of the transport machinery on peroxisomal membranes. The Arabidopsis apem (aberrant peroxisome morphology) mutant apem7 shows decreased efficiency of peroxisome targeting signal 1-dependent protein transport to peroxisomes. In apem7 mutants, peroxisome targeting signal 2-dependent protein transport is also disturbed, and plant growth is repressed. The APEM7 gene encodes a protein homologous to peroxin 4 (PEX4), which belongs to the ubiquitin-conjugating (UBC) protein family; however, the UBC activity of Arabidopsis PEX4 remains to be investigated. Here, we show using electron microscopy and immunoblot analysis using specific PEX4 antibodies and in vitro transcription/translation assay that PEX4 localizes to peroxisomal membranes and possesses UBC activity. We found that the substitution of proline with leucine by apem7 mutation alters ubiquitination of PEX4. Furthermore, substitution of the active-site cysteine residue at position 90 in PEX4, which was predicted to be a ubiquitin-conjugation site, with alanine did not restore the apem7 phenotype. Taken together, these findings indicate that abnormal ubiquitination in the apem7 mutant alters ubiquitin signaling during the process of protein transport, suggesting that the UBC activity of PEX4 is indispensable for efficient protein transport to peroxisomes.

Sucrose Production Mediated by Lipid Metabolism Suppresses the Physical Interaction of Peroxisomes and Oil Bodies during Germination of Arabidopsis thaliana.

Physical interaction between organelles is a flexible event and essential for cells to adapt rapidly to environmental stimuli. Germinating plants utilize oil bodies and peroxisomes to mobilize storage lipids for the generation of sucrose as the main energy source. Although membrane interaction between oil bodies and peroxisomes has been widely observed, its underlying molecular mechanism is largely unknown. Here we present genetic evidence for control of the physical interaction between oil bodies and peroxisomes. We identified alleles of the sdp1 mutant altered in oil body morphology. This mutant accumulates bigger and more oil body aggregates compared with the wild type and showed defects in lipid mobilization during germination. SUGAR DEPENDENT 1 (SDP1) encodes major triacylglycerol lipase in Arabidopsis Interestingly, sdp1 seedlings show enhanced physical interaction between oil bodies and peroxisomes compared with the wild type, whereas exogenous sucrose supplementation greatly suppresses the interaction. The same phenomenon occurs in the peroxisomal defective 1 (ped1) mutant, defective in lipid mobilization because of impaired peroxisomal ß-oxidation, indicating that sucrose production is a key factor for oil body-peroxisomal dissociation. Peroxisomal dissociation and subsequent release from oil bodies is dependent on actin filaments. We also show that a peroxisomal ATP binding cassette transporter, PED3, is the potential anchor protein to the membranes of these organelles. Our results provide novel components linking lipid metabolism and oil body-peroxisome interaction whereby sucrose may act as a negative signal for the interaction of oil bodies and peroxisomes to fine-tune lipolysis.