A temperature-sensitive CHO pex1 mutant with a novel mutation in the AAA Walker A1 motif.

We herein isolated a peroxisome-deficient Chinese hamster ovary mutant, ZPEG252, import-defective of peroxisome targeting signal 1 (PTS1)- and PTS2-proteins at 37 degrees C. The impaired protein import was restored at 30 degrees C, indicating a temperature-sensitive phenotype, similar to that of cells derived from patients with milder peroxisome biogenesis disorders such as infantile Refsum disease. PEX1 expression complemented the mutant phenotype of ZPEG252. Reverse transcription-PCR analysis indicated one point mutation at nucleotide residue 1817 changing a codon (GGG) for Gly(606) to a codon (GAG) for Glu(606) in the sequence for the Walker A1 motif of the AAA cassettes. This novel mutant Pex1pG606E was severely affected in binding to Pex6p at 37 degrees C, but not at 30 degrees C. Pex1pG606E was localized to peroxisomes at 30 degrees C, whilst it was discernible in a cytosolic staining pattern at 37 degrees C. Together, our findings demonstrate that Walker A1 motif of Pex1p is essential for Pex1p-Pex6p interaction and Pex1p targeting to peroxisomes.