Quantitative MS-based Proteomics Strategy to Decipher Both Stable and Transient Interactors of Membrane Proteins in Yeast Peroxisomes

摘要

Most comprehensive characterization of peroxisomal membrane protein complexes such as PeX14p, PeX25p and PeX30p using quantitative AP-MS based on SILAC. Reliable identification of "core" components and transient/weak interaction partners against a high background of co-purifying contaminants. 1. AP-AM resulted in the definition of a PeX14p core complex consisting of the entire importomer as well as two further proteins, Pex11p and Dyn2p. 2. AP-PM enabled the identification of 10 further proteins (e.g. Pex5p) transiently interacting with PeX14p or other components of the membrane core complex; most of these are so far unknown PeX14p interaction partners. Our data demonstrate that our functional proteomics strategy is a very sensitive and robust tool to identify accurately both stable core components as well as dynamically or weakly interacting proteins of low-abundant organellar membrane protein complexes in yeast. Further aim: Establishment of a most comprehensive interaction network of proteins involved in the biogenesis and proliferation of peroxisomes.