Figure 2.

Inhibition of membrane binding of pPL86 by ribosomes. (A) Microsomal membranes were preincubated with increasing amounts of mock translation mixture lacking mRNA and amino acids. In a separate tube, pPL86 was synthesized in a wheat germ system and SRP was added where indicated. The two mixtures were then combined (the numbers indicate the fold excess of the mock translation mixture over that containing mRNA). After incubation, a protease protection assay was used to determine membrane targeting of pPL86. Lane 1 shows the undigested pPL86 (total), all other samples were treated with proteinase K. Asterisk indicates the position of the ribosome-protected fragment of about 30 residues. (B) A competition experiment similar to that in A was carried out. The mock translation mixture was separated into a ribosome pellet and a cytosolic supernatant. The original mixture and both subfractions were used in a sixfold excess over mRNA-containing translation mixture in the competition experiments.