Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation
Figure 6
CADA inhibits co-translational translocation of hCD4-pPL at a post-targeting step.
(A) Schematic representation of the truncated nascent chains of [hCD4]-(7)-pPL. The nascent chains remain attached to the ribosome through a peptidyl-tRNA bond. The (approximately) 30 residues at the C-terminus of the polypeptide that are buried inside the ribosome are indicated by a grey rectangle. The SP cleavage site (at residue 25) is also marked. (B) In vitro translation, targeting, translocation (puromycin-release), and PK digestion of [35S]methionine-labelled [hCD4]-(7)-pPL 80-mers (left panel) and WT pPL 78-mers (right panel). Intact peptidyl-tRNA bands (NC-tRNA, arrow) are indicated and represent the targeted and PK protected RNCs. Note that the presence of microsomes (RM) releases some nascent chains (NC) from tRNA in an unproductive way (compare lanes 1 and 2, open arrowhead). The positions of released precursor (open arrowhead) and signal-cleaved (solid arrowhead) polypeptide chains are indicated. The SP of hCD4 but not pPL was also detected (open circle). Molecular mass is in kDa. (C) Dose response of CADA (µM) on translocation of RNCs as in (B). Graph shows translocation fractions of the autoradiogram quantified by phosphorimager analysis. (D) Time of addition of CADA. [hCD4]-(7)-pPL nascent chains of 80 residues (80-mers) were synthesized in the absence of membranes before exposure to RM for targeting. Nascent chains were left untreated (C, control), or were treated with CADA (15 µM) either administered at the beginning of the synthesis in the translation mixture (R, ribosomes), administered to membranes for pretreatment (M, microsomes), or applied to the RNC/RM mixture 15 minutes after initiation of targeting but before puromycin release (P, post-targeting). Graph shows translocation data of three experiments quantified by phosphorimager analysis. NS, not significant. *p<0.01.