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.