Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway
Fig 5
Cell free assay: activities of the compounds against the SRP-Sec61 targeting/translocation pathway.
(A) Representative digital autoradiograms of the in vitro transcribed/translated and translocated CRF1R-pPL chimaera in the absence or presence of the indicated compounds (300 μM). NCs of 78 residues without a stop codon were transcribed/translated in vitro in the cell free rabbit reticulocyte lysate system. Because of an intact peptidyl-tRNA bond, NCs stay attached to the ribosome and appear as NC-tRNA complexes (lane 1, NC-tRNA). Due to spontaneous hydrolysis of the peptidyl-tRNA bond, a fraction of the [35S]-labeled peptides is visible as prematurely released NCs (lane 1, NC). Loaded ribosomes are subsequently mixed with RM (lanes 2–5, RM) to allow for ribosome docking onto the Sec61α protein-conducting channels. Note that the presence of RM also releases extra NCs from tRNA in an unproductive way. Finally, NCs are released from the ribosomes by addition of PU (lanes 4–5, PU), resulting in signal peptide-cleaved NCs that have been translocated into the ER lumen (SPcNC, 55 residues, red arrow). In the diagram on the lower right panel, each black bar represents the relative amount of translocated protein versus total protein (translocated and precursor) after compound treatment (lane 5), in comparison to the corresponding DMSO control (= 100% translocation; lane 4). Bars are mean ± SD.; n ≥ 2. See also S1 Table for the data. P values: p < 0.0001 (***), p ≤ 0.05 (*). (B) Schematic depiction of the cell free assay following in vitro transcription/translation of construct CRF1R-pPL and addition of RM. The effects of PU treatment (right panel) or leaving the sample untreated (left panel) on the NC-tRNAs are shown. See also (A) above for details. SP = signal peptide; SPase = signal peptidase.