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

Published: 18 December 2018| Version 5 | DOI: 10.17632/w8jv9ngnsk.5
Contributors:
Richard Zimmermann,

Description

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 plus additional SP and TMH analyses, which are presented in a pptx figure and a xlsx table, respectively . Additional physicochemical properties of TRAP clients with SP or TMH. In addition to the hydrophobicity score and glycine/proline (GP) content, we determined the ΔGapp values of SP sequences (a) and transmembrane helices (TMH) (b) with the ΔGapp predictor for TM helix insertion (http://dgpred.cbr.su.se) and plotted these values against the relative count (Density). Glycine and glycine plus proline content of human signal peptides. We used custom scripts to compute the glycine (G) and glycine plus proline (GP) content of human SP sequences from UniProtKB entries. GP content was calculated as the total fraction of glycine and proline in the respective sequence. Notably, 1382 SP of a total of 3513 have a GP content of > 15%, the proposed threshold for TRAP involvement and, therefore, potential regulation by phosphorylation and/or calcium binding by TRAP subunit α.

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Institutions

Universitat des Saarlandes

Categories

Proteomics, Western Blot

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