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

Abstract

We herein isolated a peroxisome-deficient Chinese hamster ovary mutant, ZPEG252, import-defective of peroxisome targeting signal 1 (PTS1)- and PTS2-proteins at 37 °C. The impaired protein import was restored at 30 °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 °C, but not at 30 °C. Pex1pG606E was localized to peroxisomes at 30 °C, whilst it was discernible in a cytosolic staining pattern at 37 °C. Together, our findings demonstrate that Walker A1 motif of Pex1p is essential for Pex1p–Pex6p interaction and Pex1p targeting to peroxisomes.