title: Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study.
abstract: The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Wolfgang
authors: W
authors: Klein
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Carolin
authors: C
authors: Westendorf
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Antje
authors: A
authors: Schmidt
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Mercè
authors: M
authors: Conill-Cortés
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Claudia
authors: C
authors: Rutz
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Marcus
authors: M
authors: Blohs
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Michael
authors: M
authors: Beyermann
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Jonas
authors: J
authors: Protze
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Gerd
authors: G
authors: Krause
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Eberhard
authors: E
authors: Krause
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany.
authors: Ralf
authors: R
authors: Schülein
mesh_terms: D000595:Amino Acid Sequence; D051399:Aquaporin 2 / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007553:Isotope Labeling; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes / Q000737:chemistry; D010456:Peptides, Cyclic / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry
chemical_list: C496023:AQP2 protein, human; D051399:Aquaporin 2; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; C502316:cotransin