H. Otera et al., PEROXISOME TARGETING SIGNAL TYPE-1 (PTS1) RECEPTOR IS INVOLVED IN IMPORT OF BOTH PTS1 AND PTS2 - STUDIES WITH PEX5-DEFECTIVE CHO CELL MUTANTS, Molecular and cellular biology, 18(1), 1998, pp. 388-399
To investigate the mechanisms of peroxisome assembly and the molecular
basis of peroxisome assembly disorders, we isolated and characterized
a peroxisome-deficient CHO cell mutant, ZP139, which was found to bel
ong to human complementation group II, the same group as that of our e
arlier mutant, ZP105, These mutants had a phenotypic deficiency in the
import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-
terminal extension signal (PTS2)-mediated transport, including that of
3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not i
n ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated f
rom wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised
595 amino acids, 7 amino acids less than the human homolog, and conta
ined seven tetratricopeptide repeat (TPR) motifs at the C-terminal reg
ion. Chinese hamster PTS1R showed 91, 28, and 24% amino acid identity
with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae
, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues
was identified in CHO cells; for PTS1R, 37 amino acids were inserted b
etween residues at positions 215 and 216 of a shorter isoform (PTS1RS)
. Southern blot analysis of CHO cell genomic DNA suggested that these
two isoforms are derived from a single gene. Both types of PEX5 comple
mented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import
in ZP105 was rescued only by PTS1RL. This finding strongly suggests t
hat PTS1RL is also involved in the transport of PTS2. Mutations in PEX
5 were determined by reverse transcription-PCR: a G-to-A transition re
sulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of P
TS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Bo
th mutations were in the TPR domains (TPR1 and TPR6), suggesting the f
unctional consequence of these domains in protein translocation. The i
mplications of these mutations are discussed.