Proteomics explains client specificity of the human TRAP complex in ER protein import

In mammalian cells, one-third of all polypeptides are transported into/across the ER-membrane via the Sec61-channel. While the Sec61-complex facilitates translocation of all polypeptides with amino-terminal signal peptides (sp) or transmembrane helices, the Sec61-associated TRAP-complex supports translocation of only a subset, i.e. in a substrate-specific manner. To characterize TRAP-dependent precursors, we combined siRNA-mediated TRAP depletion in human cells, label-free quantitative proteomics, and differential expression analysis. The results were validated by western blotting and quantitative RT-PCR as well as under conditions of over-production of TRAP-dependent precursors and their mutated variants in the presence or absence of MG132, a proteasome inhibitor. Sp analysis of TRAP-clients revealed above-average glycine-plus-proline content as the distinguishing feature. Thus, TRAP acts as an sp-receptor on the ER-membrane’s cytosolic face for precursor polypeptides with high glycine-plus-proline content in their sp and triggers substrate-specific (and regulated) opening of the Sec61-channel by its interaction with the ER-lumenal hinge of Sec61α. The work is about to be published under the title Proteomics reveals signal peptide features determining the client specificity in human TRAP-dependent ER protein import by Duy Nguyen, Regine Stutz, Stefan Schorr, Sven Lang, Stefan Pfeffer, Hudson H. Freeze, Friedrich Förster, Volkhard Helms1 Johanna Dudek & Richard Zimmermann Here we present the original western blot data