Figure 5.

SRP-independent targeting in the reticulocyte lysate system. PPL86 was synthesized in a reticulocyte lysate system and RNCs were isolated by sedimentation through a sucrose cushion containing a low-salt concentration. One-half of the sample was treated with NEM, the other remained untreated. Before membrane targeting, low-salt-washed reticulocyte ribosomes, which were also treated with NEM, were added in increasing amounts as indicated (given as fold excess over RNCs). Membrane targeting of the RNCs was tested with a protease protection assay. Lanes 1 and 7 show the undigested pPL86 (total), all other samples were treated with proteinase K. Asterisk indicates the ribosome-protected fragment of about 30 amino acids. Arrowheads denote a fragment slightly smaller than pPL86 that is presumably protected from proteolysis by a cytosolic protein.