The peroxin Pex14p - cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional compl ementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP1 10, of complementation group 14 (CG14), The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identi ty to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex 5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) a nd PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determine d by reverse transcription-PCR as follows. A 133-base pair deletion at nucl eotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385- 487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex 14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstratin g PEX14 to be responsible for peroxisome deficiency in CG14.