SRP-dependent membrane targeting of translating ribosomes
Zusammenfassung der Projektergebnisse
Proteins of the bacterial plasma membrane are inserted into the membrane cotranslationally via the signal recognition particle (SRP) pathway. Membrane targeting of ribosomes translating membrane proteins is initiated by recmiting SRP to translating ribosomes. According to our crosslinking results, the binding site of SRP is located at the back of the large ribosomal subunit where the nascent peptide emerges from the exit tunnel. The binding site comprises protein L23 which is located at the tunnel exit and extends a loop into the tunnel. Furthermore, we could show that SRP and trigger factor, which share protein L23 as (part of) their binding sites, can be bound to the same ribosome simultaneously. Only when the targeting complex is completed by binding ofthe SRP receptor, FtsY, trigger factor is displaced. Unexpectedly, and in contrast to the classic targeting model, we found that SRP initially is recruited to all translating ribosomes, independent of the exposure of a signal-anchor sequence (SAS) on the outside of the ribosome, as long as the nascent peptide is contained within the tunnel. Compared to vacant or non-translating ribosomes, the presence of the nascent peptide within the tunnel increases the affinity of SRP binding nearly 100-fold (to <1 nM). The intratunnel loop of protein L23 is important for signaling the presence of the nascent peptide to the outside. Thus, the classic model had to be extended to include an early targeling step that was independent of the presence of an SAS in the nascent peptide. The interaction with the ribosome induces a conformation of SRP in which the NG domain of Ffh is fully exposed for the high-affinity interaction with FtsY. When the growing peptide appears at the outside of the ribosome and exposes an SAS, the high-affinity targeting complex persists, and the interaction of the translating ribosome with the translocon via binding to FtsY is established. Exposure of a non-SAS sequence abolishes SRP binding and releases the translating ribosomes back into the cytosol. We propose that this mechanism of early indiscriminate membrane targeting lengthens the time window for bringing translating ribosomes to the membrane, thereby increasing the chance of proper membrane targeting of ribosomes synthesizing membrane proteins, which in the cytosol tend to aggregate and harm the cell. This may alleviate the lack of translational stalling in prokaryotes, which in eukaryotes halts translation until ribosomes synthesizing proteins for membrane insertion or export are properly targeted to the translocon. There is very little SRP relative to ribosomes, probably less than five percent. Thus, the model of initial indiscriminate recruitment of SRP to all translating ribosomes, which is suggested by our results, requires that SRP rapidly samples all ribosomes until ribosomes synthesizing a membrane protein are identified by an exposed SAS. In fact, the kinetic analyses we have performed show that SRP dissociates rapidly from vacant or translating ribosomes, unless an SAS is exposed on the ribosome, which slows down dissociation considerably. Interestingly, the same effect is observed when the SAS peptide is added separately, indicating that SAS binding induces a conformation of SRP that binds to the ribosome, as well as to the SRP receptor FtsY, with high stability. Based on FRET measurements, the conformational change induced by ribosome binding entails an opening of the Ffh structure in that NG and M domains move apart, while the conformational change induced by signal peptide binding seems to be confined to the M domain. The interaction of the ribosome-SRP-FtsY complex with the translocon remains to be studied in detail. To obtain the translocon in a biochemically better defined form, we have adopted the method to incorporate the translocon into lipoprotein-like particles of defined size ("nanodiscs"), rather than using proteoliposomes, which are not very well defined and are of low activity. The interactions between nanodisc-embedded translocons, the SRP receptor FtsY, and the ribosome-SRP complex are currently being studied. Another problem which could not be solved yet is the timing of GTP hydrolysis on both SRP and FtsY which is expected to initiate the disassembly of the targeting complex that accompanies the transfer of the translating ribosome fi-om SRP to the translocon. Finally, the conformational changes of the translocon that presumably take place when a ribosome exposing a signal-anchor sequence binds will be characterized in future work on this project.
Projektbezogene Publikationen (Auswahl)
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(2008). Signal sequenceindependent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel. Nature Struct Mol. Biol. 15, 494-499
Bornemann, T., Jöckel, J., Rodnina, M. V., Wintermeyer, W.
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(2009). Conformation of the signal recognition particle in ribosomal targeting complexes. RNA 15, 44-54
Buskiewicz, I.A., Jöckel, J., Rodnina, M.V., Wintermeyer, W.
