title: Identification of core components and transient interactors of the peroxisomal importomer by dual-track stable isotope labeling with amino acids in cell culture analysis.
abstract: 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.
authors.affiliation: Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Oeljeklaus
authors.forename: Benedikt S
authors.initials: BS
authors.lastname: Reinartz
authors.forename: Janina
authors.initials: J
authors.lastname: Wolf
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wiese
authors.forename: Jason
authors.initials: J
authors.lastname: Tonillo
authors.forename: Katharina
authors.initials: K
authors.lastname: Podwojski
authors.forename: Katja
authors.initials: K
authors.lastname: Kuhlmann
authors.forename: Christian
authors.initials: C
authors.lastname: Stephan
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schliebs
authors.forename: Cécile
authors.initials: C
authors.lastname: Brocard
authors.forename: Ralf
authors.initials: R
authors.lastname: Erdmann
authors.forename: Bettina
authors.initials: B
authors.lastname: Warscheid
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D007553:Isotope Labeling / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D020675:Peroxisomes / Q000737:chemistry* / Q000378:metabolism; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D012107:Research Design; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins