Tomoyuki Harano

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 complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha andSaccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity 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 Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors’ cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide 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 Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Hsp70 regulates the interaction between the peroxisome targeting signal type 1 (PTS1)-receptor Pex5p and PTS1

The peroxisome targeting signal type 1 (PTS1) receptor, Pex5p, of the tetratricopeptide repeat (TPR) motif family is located mostly in the cytosol and mediates the translocation of PTS1 proteins to peroxisomes. As a step towards understanding the mechanisms of protein import into peroxisomes, we investigated the molecular mechanisms involved in PTS1 recognition by Pex5p with regard to requirement of energy and cytosolic factors, using cell-free synthesized acyl-CoA oxidase (AOx) as a PTS1 cargo protein, together with Pex5p and heat-shock protein (Hsp)70 from rat liver. Pex5p was partly associated with peroxisomes of rat liver, was resistant to washing with a high concentration of salt and to alkaline extraction and was inaccessible to protease added externally. Pex5p bound to AOx in an ATP-dependent manner. AOx synthesized in a cell-free translating system from rabbit reticulocyte lysate was imported into peroxisomes without being supplemented with Pex5p and Hsp70, implying that peroxisome-associated Pex5p was released from the membranes and functional in this in vitro import assay. Antibodies against Pex5p and Hsp70 inhibited AOx import. In contrast, AOx synthesized in a wheat-germ lysate required the external addition of Pex5p for import, in which Hsp70 augmented the AOx import. The TPR domain of Pex5p was revealed to bind to the N-terminal part in an Hsp70-independent manner, whereas mutual interaction of the TPR region was noted in the presence of Hsp70. Hsp70 interacted with the TPR domain of Pex5p. Moreover, Hsp70 and ATP synergistically enhanced the binding of Pex5p to the C-terminal PTS1-containing part of AOx, implying that Pex5p recognizes its cargo PTS1 protein by chaperone-assisted as well as energy-dependent mechanisms in vivo.

Presence of two different types of protein-disulfide isomerase on cytoplasmic and luminal surfaces of endoplasmic reticulum of rat liver cells

Summary The presence of two types of protein-disulfide isomerase in rat liver microsomes was demonstrated by their different susceptibility to detergent treatment. The activity detectable with intact microsomes was strongly inhibited by low concentrations of Triton X-100. Higher concentrations of the detergent caused significant stimulation of the enzyme activity, which was latent in intact microsomes. The detergent-labile enzyme was purified from the acetone powder of microsomes. Judging from the effect of the antibody, which was prepared against the purified enzyme, on the enzyme activities of intact and detergent-treated microsomes, the detergent-labile enzyme is located on the cytoplasmic surface of endoplasmic reticulum, whereas another type of the enzyme, which is latent in intact microsomes, is located on the luminal surface.