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.