Identification of core components and transient interactors of the peroxisomal importomer by dual-track stable isotope labeling with amino acids in cell culture analysis.

The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer.

Peroxisomal targeting of PTS2 pre-import complexes in the yeast Saccharomyces cerevisiae.

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.

Long- and short-haired Weimaraner dogs represent two populations of one breed.

Weimaraners represent an old breed of hunting dogs. Today, two coat types are commonly distinguished, the more common short-hair (SH) and the long-hair (LH) variety, the latter having arisen from the SH Weimaraners. In order to analyze genetic variation in the coat varieties, we genotyped nine single nucleotide polymorphisms (SNPs) of the ABCA4 gene locus as well as six highly variable microsatellites scattered over the canine genome in the SH and LH populations. Three out of nine SNPs showed two alleles, allelic frequencies at two of these polymorphic sites differed significantly between SH and LH Weimaraners. Haplotype diversities for the three informative SNPs revealed higher estimates for the SH (0.515) than for the LH variety (0.364). In addition, two of six microsatellite markers showed significant differences in allelic frequencies between SH and LH Weimaraners. Unexpectedly, genetic diversities for all but one microsatellite were greater in LH than in SH Weimaraners. Similarly, the mean intra-individual genetic distance based on microsatellite markers was more pronounced in the LH population (0.62 for SH vs. 0.65 for LH) suggesting again closer genetic relationships among SH than LH Weimaraners. Taken together, the results of SNP analysis can be interpreted as reflections of early breed development whereas microsatellites mirror rather recent breeding strategies in the Weimaraner populations.