Analyses of the dynamic interplay between ribosomes and bacterial translocon complexes
Final Report Abstract
The recognition and transport of proteins destined to reside in different cellular compartments is an inherent and essential process in eukaryotic and prokaryotic cells. Although a large number of different protein transport pathways have been identified in different species, only a few pathways appear to be universally conserved. An example is the Signal recognition particle (SRP) dependent protein transport to the Sec translocon, a membrane embedded protein transport channel. SRP recognizes the signal sequence of its substrates co-translationally and the ribosomal protein L23 has been identified as a major docking site for SRP. However, even when a signal sequence is still within the ribosomal tunnel and not yet exposed to the outside, its presence enhances SRP binding to the ribosome. This suggests that the presence of a signal sequence inside the ribosomal tunnel is communicated to the tunnel exit, resulting in the subsequent recruitment of targeting factors. Our data show that SRP and SecA, the motor protein of the post-translational targeting pathway in bacteria, are able to contact a flexible loop of L23 which is located inside the ribosomal tunnel. This offers a possible explanation for high-affinity binding of SRP to translating ribosomes before the signal sequence is fully exposed to the outside of the tunnel. The data furthermore suggest that competition between the SecA- and SRP-dependent protein targeting pathways is already initiated at the ribosome. Targeting to and insertion via the Sec translocon depends on an intricate protein-protein interaction network and determining these interactions in resting and actively transporting Sec translocons has been a major accomplishment during the funding period. This was achieved by using a site-directed photo cross-linking approach both in vivo and in vitro. In combination with mass spectrometry, we determined the exact interaction surface of the known partner proteins YidC and FtsY, the bacterial SRP receptor. We also identified new partner proteins, like the periplasmic chaperone PpiD. The physiological significance of these interactions was further studied, which allowed us to propose a new model for the transfer of ribosome-nascent chains from the SRP targeting machinery to the Sec channel. We furthermore determined the influence of YidC on the Sec channel geometry and found a reduced channel cross section and contacts of YidC with the channel interior. These unexpected results suggest that YidC is at least transiently part of the channel surface. In bacteria, membrane proteins are not only membrane inserted via the Sec translocon but also via YidC, which was shown to insert small membrane proteins independently of the Sec translocon. However, the determinants that route membrane proteins either into the Sec pathway or the YidC pathway were unknown. Our data showed that multi-spanning membrane proteins like MtlA or TatC can also be inserted via YidC. Only membrane proteins with large periplasmic domains seem to be exclusively Sec dependent. This is most likely related to the fact that the translocation of periplasmic domains depends on SecA, which can interact with the Sec translocon but not with YidC. These multi-spanning membrane proteins are strictly SRP/FtsY dependent and our data suggest that the SRP pathway promiscuously delivers membrane proteins to either the Sec translocon or YidC for insertion. Interaction of YidC with SRP, FtsY and ribosomal proteins was demonstrated by cross- linking experiments. Electrophysiological data furthermore indicate that YidC forms like the Sec translocon a protein conducting channel.
Publications
- (2008). A cleavable N- terminal membrane anchor is involved in membrane binding of the Escherichia coli SRP receptor. J. Mol. Biol. 377, 761-773
Weiche, B., Bürk, J., Angelini, S., Schiltz, E., J.O. Thumfart und Koch, H.G.
(See online at https://doi.org/10.1016/j.jmb.2008.01.040) - (2009) Predominant membrane localization is an essential feature of the bacterial SRP receptor. BMC Biology, 7, 76
Mircheva, M., Boy, D., Weiche, B., Hucke, F., Graumann, P., und Koch, H.G.
(See online at https://doi.org/10.1186/1741-7007-7-76) - (2009) The depletion of the SRP receptor inactivates ribosomes in Escherichia coli. J. Bact. 191, 7017-7026
Bürk, J., Weiche, B., Wenk, M., Boy, D., Nestel, S., Heimrich, B., und Koch, H.G.
(See online at https://doi.org/10.1128/JB.00208-09) - (2009) Two cooperating helices constitute the lipid binding domain of the bacterial SRP receptor. J. Mol. Biol. 390, 401-413
Braig, D., Bär C., Thumfart, J.O., und Koch, H.G.
(See online at https://doi.org/10.1016/j.jmb.2009.04.061) - (2009). Visualization of distinct entities of the SecYEG translocon during translocation and integration of bacterial proteins. Mol. Biol. Cell 20, 1804-1815
Boy, D. und Koch, H.G.
(See online at https://doi.org/10.1091/mbc.e08-08-0886) - (2011). Signal-sequence independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle. Mol. Biol. Cell 22, 2309-2323
Braig, D., Mircheva, M., Sachelaru, I., van der Sluis, E.O., Sturm, L., Beckmann, R., und Koch, H.G.
(See online at https://doi.org/10.1091/mbc.e11-02-0152) - (2011). The bacterial SRP receptor, SecA and the ribosome use overlapping binding sites on the SecY translocon. Traffic 12, 563-578
Kuhn, P., Weiche, B., Sturm, L., Sommer, E., Drepper, F.,Warscheid, B., Sourjik, V., und Koch, H.G.
(See online at https://doi.org/10.1111/j.1600-0854.2011.01167.x) - (2012) The universally conserved ATPase YchF acts as an inhibitor of the oxidative stress response. J. Biol. Chem., 287, 43585-43598
Wenk, M., Ba, Q., Erichsen, V., MacInnes, K., Wiese, H., Warscheid, B., and Koch, H.G.
(See online at https://doi.org/10.1074/jbc.M112.413070) - (2012). Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the E. coli Signal recognition particle. Mol. Biol. Cell 23. 464-479
Welte, T., Kudva, R., Kuhn, P., Sturm, L., Braig, D., Müller, M., Warscheid, B., Drepper, F., and Koch, H.G.
(See online at https://doi.org/10.1091/mbc.E11-07-0590) - (2013). Protein translocation across the inner membrane of Gram-negative bacteria: The Sec and Tat dependent protein transport pathways. Research in Microbiology 164; 505-534. Special Issue on Bacterial protein secretion systems (C. Wandersman, ed.)
Kudva, R., Denks, K., Kuhn, P., Vogt,, A., Müller, M and Koch, H.G.
(See online at https://doi.org/10.1016/j.resmic.2013.03.016) - (2013). YidC occupies the lateral gate of the SecYEG translocon and is sequentially displaced by a nascent membrane protein. J. Biol. Chem. 288, 16295-16307
Sachelaru, I., Petriman, N.A., Kudva, R., Kuhn, P., Welte, T., Knapp, B., Drepper, F., Warscheid, B., and Koch, H.G.
(See online at https://doi.org/10.1074/jbc.M112.446583) - (2014) Dynamic interaction of the Sec translocon with the periplasmic chaperone PpiD. J. Biol. Chem.
Sachelaru, I., Petriman, N.A., Kudva, R. and Koch, H.G.
(See online at https://doi.org/10.1074/jbc.M114.577916) - (2014) Signal recognition particle and SecA cooperate during export of secretory proteins with highly hydrophobic signal sequences. PLoS ONE 9: e92994
Zhou, Y., Ueda, T. and Müller, M.
(See online at https://doi.org/10.1371/journal.pone.0092994) - (2014). Targeting and integration of bacterial membrane proteins. In: Bacterial Membranes: Structural and Molecular Biology (Remaut, H., and Fronzes, R. eds). Horizon Press, Norwich, U.K.
Kuhn, P., Kudva, R., Welte, T., Sturm, L., and Koch, H.G.
- (2014). The Sec translocon mediated protein transport in prokaryotes and eukaryotes. Mol. Membrane Biology, 31, 58-84
Denks, K., Vogt, A.; Sachelaru, I., Petriman, N.A. Kudva, R. and Koch, H.G.
(See online at https://doi.org/10.3109/09687688.2014.907455)
