We earlier isolated peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, ZPEG241, by the 9-(1′-pyrene)nonanol/ultraviolet selection method, from TKaEG2, the wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal type 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). Peroxisomal localization of PTS2-EGFP was specifically impaired in ZPEG241 due to the failure of Pex5pL expression. Analysis of partial genomic sequence of PEX5 revealed one-point nucleotide-mutation from G to A in the 3′-acceptor splice site located at 1 nt upstream of exon 7 encoding Pex5pL specific 37-amino acid insertion, thereby generating 21-nt deleted mRNA of PEX5L in ZPEG241. When ZPEG241-derived Pex5pL was ectopically expressed in ZPEG241, PTS2 import was not restored because of no interaction with Pex7p. Together, we confirm the pivotal role of Pex5pL in PTS2 import, showing that the N-terminal 7-amino acid residues in the 37-amino acid insertion of Pex5pL are essential for the binding to Pex7p.
All Science Journal Classification (ASJC) codes
- Molecular Biology