FIGURE 7:

NAC is required for the recruitment of SRP when the SA segment is localized inside the ribosomal tunnel. (A) ³⁵S-Labeled RNCs generated in wild-type, Δegd1Δegd2, or Δsrp54 translation extract were allowed to bind to high-salt-washed microsomal membranes. After reisolation, microsomal membranes and bound RNCs were run on Tris-tricine gels and were analyzed via autoradiography. The total represents 25% of Pgk1-120 or Dap2-120-RNCs added to high-salt-washed microsomes. (B) FLAG–nascent chain pull downs with RNCs carrying Dap2∆-62 (Supplemental Figure S2). RNCs were generated in Δegd1Δegd2 or ∆srp54 translation extract to which wild-type extract containing NAC and SRP was added after inhibition of translation. The amount of wild-type extract corresponded to 1× or 2× the volume of the Δegd1Δegd2 or a ∆srp54 translation extract used in the translation reaction. FLAG–nascent chain pull downs (Supplemental Figure S3B) were then performed and were analyzed as described in Figure 1. (C) RNCs carrying untagged Dap2-60 or Dap2-120 were generated in a FLAG-Egd2 translation extract. Subsequently FLAG-NAC pull downs (Supplemental Figure S3C) were performed and were analyzed as in Figure 4. (D) RNCs carrying FLAG-tagged (FLAG +) or untagged (FLAG –) Dap2-60 were generated in wild-type or Δegd1Δegd2 translation extract. As indicated, the Δegd1Δegd2 translation extract was complemented with a final concentration of 1 μM purified NAC (+ NAC) before the translation reaction.