Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import

We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis, The pex12 Delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway. The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids. ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S, cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus. Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol, Pex12p - deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12 Delta null mutant cells. This finding indicates that pex12 Delta cells are not impaired in peroxisomal membrane biogenesis. In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p, Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex1op-Pex12p interaction. Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.</p

Peroxisomes:organelles at the crossroads

Recent years have seen remarkable progress in our understanding of the function of peroxisomes in higher and lower eukaryotes. Combined genetic and biochemical approaches have led to the identification of many genes required for the biogenesis of this organelle. This review summarizes recent, rather surprising, results and discusses how they can be incorporated into the current view of peroxisome biogenesis

The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene

The import of peroxisomal matrix proteins is dependent on one of two targeting signals, PTS1 and PTS2. We demonstrate in vivo that not only the import of thiolase but also that of a chimeric protein consisting of the thiolase PTS2 (amino acids 1-18) fused to the bacterial protein β-lactamase is Pas7p dependent. In addition, using a combination of several independent approaches (two-hybrid system, co-immunoprecipitation, affinity chromatography and high copy suppression), we show that Pas7p specifically interacts with thiolase in vivo and in vitro. For this interaction, the N-terminal PTS2 of thiolase is both necessary and sufficient. The specific binding of Pas7p to thiolase does not require peroxisomes. Pas7p recognizes the PTS2 of thiolase even when this otherwise N-terminal targeting signal is fused to the C-terminus of other proteins, i.e. the activation domain of Gal4p or GST. These results demonstrate that Pas7p is the targeting signal-specific receptor of thiolase in Saccharomyces cerevisiae and, moreover, are consistent with the view that Pas7p is the general receptor of the PTS2. Our observation that Pas7p also interacts with the human peroxisomal thiolase suggests that in the human peroxisomal disorders characterized by an import defect for PTS2 proteins (classical rhizomelic chondrodysplasia punctata), a functional homologue of Pas7p may be impaired

Fate of linoleic, arachidonic, and docosa-7,10,13,16-tetraenoic acids in rat testicles

A comparative study was made on the fate of linoleic, arachidonic, and docosa-7,10,13,16-tetraenoic acids in various subcellular fractions of liver and testis from rats of different ages. It was demonstrated that testicular microsomes can desaturate and elongate linoleic and arachidonic acids in a manner similar to liver microsomes, and that testicular mitochondria can convert docosa-7,10,13,16-tetraenoic acid to arachidonic acid. Testicular or liver microsomes actively desaturate linoleic acid to γ-linolenic acid and eicosa-8,11,14-trienoic acid to arachidonic acid. However, it was impossible to measure in vitro any direct conversion of adrenic acid (22:4 [n – 6]) to docosapentaenoic acid (22: 5 [n – 6]) by either liver or testicular microsomes. Docosa-7,10,13,16-tetraenoic acid is incorporated preferentially into the triglyceride fraction of total testis, mitochondria, and microsomes, while linoleic and arachidonic acids are incorporated more into phospholipids. The capacity of testicular microsomes, but not of liver microsomes, to synthesize polyunsaturated fatty acids declines with age. It is proposed that the synthesis of acids of the linoleic family proceeds in two stages, a rapid one in which arachidonic acid is made and a second, slower, one in which C22 and C24 acids are synthesized. In addition, there appears to be a cycle between microsomes and mitochondria that acts to conserve essential polyunsaturated C20 and C22 fatty acids by means of synthesis and partial degradation, respectively. This cycle would restrict the loss of essential fatty acids and might be of importance for the supply of arachidonic acid in testis under specific requirements and especially in older animals.Facultad de Ciencias Médica