No abstract

In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisomedeficient 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 and Saccharomyces 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 Cterminal 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.

Mandelonitrile benzoate, a minor defense component produced by polydesmoid millipedes, is produced in large amounts together with hydrogen cyanide following shake-disturbances administered to individuals of Nedyopus tambanus tambanus, Parafontaria tonominea, Epanerchodus sp., and Epanerchodus fulvus. These species commonly produce mandelonitrile and benzoyl cyanide (the oxidized product after discharge). The newly generated mandelonitrile benzoate was identified as a product of post secretion Schotten-Baumann reaction under basic conditions of bled bodily fluids (pH ca. 9.0), and was not an enzymatic reaction product. The reaction occurred in vitro even under less basic conditions [1M Tris-HCl buffer (pH 8.0)], and could be defined as a new mechanism of hydrogen cyanide release occurring in roughly half of polydesmoid millipedes. Species possessing no benzoyl cyanide, such as Oxidus gracilis and Cryptocorypha sp., could also produce mandelonitrile benzoate under conditions in which benzoyl cyanide was exogenously provided.

The female sex pheromone of Rhizoglyphus setosus Manson (Astigmata: Acaridae) was identified as S-isorobinal (4S-4-isopropenyl-3-oxo-1-cyclohexene-1-carboxyaldehyde), which stimulated males sexually and enhanced the frequency of the male's tapping and mounting behavior. Although the female hexane extract indicated no sign of sex pheromone activity against tested males, possibly due to the presence of the alarm pheromone neryl formate, an SiO2 column fraction containing isorobinal elicited sex pheromone activity at a dose of one female equivalent. The stereochemistry of natural isorobinal was identified as S by an HPLC using a chiral column. Both S- and R-isorobinals exhibited maximum activity at the same dose of 1 and 10 ng with a convex dose-response relationship. Amounts of S-isorobinal were determined to be 11.7 +/- 1.0 ng per female and 6.4 +/- 1.3 ng per male by GLC. This is the second example of two pheromones (the alarm pheromone neryl formate, and the sex pheromone S-isorobinal) demonstrated to be components of the same opisthonotal gland secretion.