Figure 1.

NAC prevents targeting by blocking the interaction of ribosomes with the Sec61 complex. High salt-stripped 77aaffLuc RNCs were prepared in a reticulocyte lysate translation system supplemented with ^[35]S-Met. RNCs at the final concentration of 3 OD₂₆₀/ml were incubated with 1.5 μM NAC or NAC buffer for 2 min at 26°C and 5 min on ice before the addition of 1 equivalent (eq) of puromycin/KOAc-washed microsomes (PKRMs) (30) or reconstituted Sec61 complex containing proteoliposomes (17) as indicated. Total assay volumes were 20 μl. Binding was assessed with the flotation assay. Bound RNCs are recovered in top (T) fractions whereas free RNCs are in bottom (B) fractions. In vitro transcription and translation of truncated mRNAs were as described (12) for 20 min at 26°C. Salt stripping and sedimentation of RNCs was as described (5). The complexes were resuspended in blank buffer lacking nucleotides and energy-generating systems unless otherwise indicated. RNC binding was assayed as described (14). The content of radiolabeled nascent chains in each fraction was analyzed by SDS/PAGE and fluorography or scintillation counting.