Signal sequence–independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel

Abstract

Ribosomes synthesizing inner membrane proteins in Escherichia coli are targeted to the translocon in the plasma membrane by the signal recognition particle (SRP) and the SRP receptor, FtsY. Here we show using a purified system that membrane targeting does not require an exposed signal-anchor sequence, as SRP-dependent targeting takes place with ribosomes containing short nascent peptides, with or without a signal-anchor sequence, within the peptide exit tunnel. Signaling from inside the tunnel involves ribosomal protein L23, which constitutes part of the SRP binding site. When nascent peptides emerge from the ribosome, the targeting complex is maintained with ribosomes exposing a signal-anchor sequence, whereas ribosomes exposing other sequences are released. These results indicate that ribosome-nascent chain complexes containing any nascent peptide within the exit tunnel can enter the SRP targeting pathway to be sorted at the membrane into ribosome-nascent chain complexes that synthesize either membrane or cytosolic proteins.

Interplay between trigger factor and other protein biogenesis factors on the ribosome

“…Furthermore, the fitting yielded intrinsic K d values for the binding of TF, around 50 nM, and SRP, around 80 nM, consistent with the values obtained by direct titration in the absence of competitor (Supplementary Fig. 2; Bornemann et al 12 ). Finally, the evaluation of the titrations yielded an intrinsic K d of PDF binding to non-translating ribosomes of around 1.5 mM, comparable to a published value 28 .…”

“…Ribosomes from E. coli MRE600, initiation factors IF1, IF2 and IF3, EF-Tu, EF-G, f[ 3 H]Met-tRNA fMet and total tRNA were prepared following standard procedures 39 . RNCs were prepared by in vitro translation of truncated mRNAs and purified by sucrose gradient centrifugation 12 . About 90% of the ribosomes carried the respective peptide chain.…”

“…In contrast, SRP is recruited to translating ribosomes early, even before the nascent peptide emerges from the exit tunnel 12 , and forms a rather stable complex (half-life time about 0.5 s, ref. 13).…”

“…In the 'scanning' mode, SRP binds to non-translating ribosomes or ribosomes that do not synthesize membrane proteins in a readily reversible fashion 13 , with effective dissociation rates in the range of 10 s À 1 , allowing other RPBs to bind in a stochastic fashion. However, the extent to which RPBs can enter when SRP is stabilized on translating ribosomes with short nascent chains ('stand-by'; effective off-rate about 1 s À 1 ) or on ribosomes synthesizing membrane proteins ('targeting'; effective off-rate about 0.1 s À 1 ) 12,13 is not clear.…”

“…SRP scans translating ribosomes for emerging hydrophobic signal-anchor sequences (SAS). Binding to an SAS switches SRP to the targeting mode and initiates cotranslational membrane targeting by binding to the SRP receptor and transfer to the translocon 12,13 . SRP and TF together can bind to the ribosome, although both contact protein L23 at the peptide exit ( Fig.…”