Structural dynamics and regulation of the Sec61 complex
Final Report Abstract
The focus of the joint project was the mammalian Sec61 complex, in particular, its dynamics and regulatory-components and -mechanisms. The heterotrimeric Sec61 complex resides in the membrane of the endoplasmic reticulum (ER) and can form an aqueous channel for membrane insertion and translocation of nascent and newly-synthesized precursor polypeptides. Since the Sec61 channel is also permeable to calcium ions and the ER is the major calcium storage organelle of mammalian cells, the opening and closing, i.e. the gating, of the Sec61 complex has to be tightly controlled. In our original collaborative effort, single channel recordings from purified and reconstituted canine pancreatic Sec61 complexes had characterized the complex as a dynamic precursor polypeptide gated channel. In the course of this joint project, the dynamic behaviour of the Sec61 channel was found to be essential for its protein transport activity and also observed for the ortholog yeast Sec61 complex. Furthermore, the ER lumenal Hsp70, BiP, and the cytosolic calmodulin were identified as regulatory components of the mammalian Sec61 channel, which induce the gating to the closed state. This led to the hypothesis that BiP binding to the Sec61 channel is involved in limiting Ca2+ leakage from the ER at the level of the Sec61 complex and that, after Ca2+ has started to leak out, Ca2+ binding leads to recruitment of Ca2+-calmodulin to the channel and subsequent channel closure. This hypothesis was fully confirmed by live cell Ca2+ imaging experiments with human cells. Furthermore, this approach identified the ER membrane protein Sec62 as an additional player in Sec61 channel closure and characterized ER lumenal loop 7 of Sec61α, a cytosolic IQ- motif in the aminoterminal region of Sec61, and the aminoterminus of Sec61α as binding sites for BiP, Ca2+-calmodulin, and Sec62, respectively. These binding sites were confimed and quantified by fluorescence correlation spectroscopy, peptide spot arrays, and surface plasmon resonance spectroscopy. Various bioinformatic analyses were also consistent with these observations and allowed us to suggest an atomic model for the gating of the mammalian Sec61 channel by its three allosteric effectors. Recently, this model was incorporated into a bigger picture, which suggests that the Ca2+ permeability of the mammalian Sec61 complex actually serves a function under ER stress conditions, i.e. allows prolonged Ca2+ efflux from the ER, i.e. when BiP is sequestered by unfolded polypeptide chains, for the shift from the unfolded stress response to apoptosis. We note that this concept is consistent with our observation that a mutant Sec61 complex, which cannot be bound and regulated by BiP, leads to apoptosis of pancreatic ß-cells and diabetes in mice. We coined the term Sec61-channelopathies for this kind of diseases. With respect to protein biogenesis at the mammalian ER, we observed that Ca2+-calmodulin selectively inhibits membrane insertion of tail-anchored membrane proteins, while not affecting ER import of nascent precursors of secretory or plasma membrane proteins. Furthermore, we observed that interaction of BiP with loop 7 is required for insertion of certain precursor polypeptides into the Sec61 complex, i.e. can also be involved in gating of the Sec61 channel to the open state. This raises the question how BiP can gate the channel from closed to open and vice versa. We are convinced that the presence or absence of the precursor polypeptide is crucial and that the co- chaperones play a role
Publications
- (2009) A novel twist to protein secretion in eukaryotes. Trends in Parasitology 25: 147-150
Zimmermann, R., Blatch, G.L.
(See online at https://doi.org/10.1016/j.pt.2009.01.002) - (2009) Lanthanum inhibits the mammalian Sec61 complex in its channel dynamics and protein transport activity. FEBS Lett. 583: 2359-2364
Erdmann, F., Jung, M., Eyrisch, S., Lang, S., Helms, V., Wagner, R., Zimmermann, R.
(See online at https://doi.org/10.1016/j.febslet.2009.06.032) - (2010) Characterization of horizontal lipid bilayers as a model system to study lipid phase separation. Biophys. J. 98: 1-9
Honigmann, A., Walter, C., Erdmann, F., Eggeling, C., Wagner, R.
(See online at https://doi.org/10.1016/j.bpj.2010.03.033) - (2010) Evolutionary gain of function of the ER membrane protein Sec62 from yeast to humans. Mol. Biol. Cell 21: 691-703
Müller, L., Diaz de Escauriaza, M., Lajoie, P., Theis, M., Jung, M., Müller, A., Burgard, C., Greiner, M., Snapp, E.L., Dudek, J., Zimmermann, R.
(See online at https://doi.org/10.1091/mbc.e09-08-0730) - (2010) Protein conducting nanopores. J. Phys.: Condens. Matter 22: 454102-454122
Harsman, A., Krüger, V., Bartsch, P., Honigmann, A., Schmidt, O., Rao, S., Meisinger, C., Wagner, R.
(See online at https://doi.org/10.1088/0953-8984/22/45/454102) - (2010) The mammalian and yeast translocon complexes comprise a characteristic Sec61 channel. Biochem. Biophys. Res. Commun. 396: 714-720
Erdmann, F., Jung, M., Maurer, P., Harsmann, A., Zimmermann, R., Wagner, R.
(See online at https://doi.org/10.1016/j.bbrc.2010.04.168) - (2011) Calcium-calmodulin inhibits tail-anchored protein insertion into the mammalian endoplasmic reticulum membrane. FEBS Lett. 585: 3485-3490
Haßdenteufel, S., Schäuble, N., Cassella, P., Leznicki, P., Müller, A., High, S., Jung, M., Zimmermann, R.
(See online at https://doi.org/10.1016/j.febslet.2011.10.008) - (2011) Calmodulin regulation of the calcium-leak channel Sec61 is unique to vertebrates. Channels 5: 293-298
Harsman, A., Kopp, A., Wagner, R., Zimmermann, R., Jung, M.
(See online at https://doi.org/10.4161/chan.5.4.16160) - (2011) Exploring protein import pores of cellular organelles at the single molecule level using the planar lipid bilayer technique. Eur. J. Cell Biol. 90: 721-730
Harsman, A., Bartsch, P., Hemmis, B., Krüger, V., Wagner, R.
(See online at https://doi.org/10.1016/j.ejcb.2011.04.012) - (2011) Interaction of calmodulin with Sec61a limits Ca2+ leakage from the endoplasmic reticulum. EMBO J. 30: 17-31
Erdmann, F., Schäuble, N., Lang, S., Jung, M., Honigmann, A., Ahmad, M., Dudek, J., Benedix, J., Harsmann, A., Kopp, A., Helms, V., Cavalié, A., Wagner, R., Zimmermann, R.
(See online at https://doi.org/10.1038/emboj.2010.284) - (2011) Protein translocation across the ER membrane. Biochim. Biophys. Acta 1808: 912-924
Zimmermann, R., Eyrisch, S., Ahmad, M., Helms, V.
(See online at https://doi.org/10.1016/j.bbamem.2010.06.015) - (2011) Sec61 complexes form ubiquitous ER Ca2+ leak channels. Channels 5: 228-235
Lang, S., Erdmann, F., Jung, M., Wagner, R., Cavalié, A., Zimmermann, R.
(See online at https://doi.org/10.4161/chan.5.3.15314) - (2012) BiP-mediated closing of the Sec61 channel limits Ca2+ leakage from the ER. EMBO J. 31: 3282-3296
Schäuble, N., Lang, S., Jung, M., Cappel, S., Schorr, S., Ulucan, Ö., Linxweiler, J., Dudek, J., Blum, R., Helms, V., Paton, A. W., Paton, J. C., Cavalié, A., Zimmermann, R.
(See online at https://doi.org/10.1038/emboj.2012.189) - (2012) Horizontal bilayer for electrical and optical recordings. Materials 5: 2705-2730
Bartsch, P., Walter, C., Selenschik, P., Honigmann, A., Wagner, R.
(See online at https://doi.org/10.3390/ma5122705) - (2012) Structure and 3D arrangement of ER-membrane associated ribosomes. Structure 20: 1508-1518
Pfeffer, S., Brandt, F., Hrabe, T., Lang, S., Eibauer, M., Zimmermann, R., Förster, F.
(See online at https://doi.org/10.1016/j.str.2012.06.010) - (2013) Single channel analysis of membrane proteins in artificial bilayer membranes. Methods Mol. Biol. 1033: 345-361
Bartsch, P., Harsman, A., Wagner, R.
(See online at https://doi.org/10.1007/978-1-62703-487-6_22) - (2013) Targeting cell migration and the ER stress response with calmodulin antagonists: A clinically tested small molecule phenocopy of SEC62 gene silencing in human tumor cells. BMC – cancer 13: 574
Linxweiler, M., Schorr, S., Jung, M., Schäuble, N., Linxweiler, J., Langer, F., Schäfers, H.-J., Cavalié, A., Zimmermann, R., Greiner, M.
(See online at https://doi.org/10.1186/1471-2407-13-574) - (2013) The signal sequence influences posttranslational ER translocation at distinct stages. PLOS ONE 8: e75394
Johnson, N., Haßdenteufel, S., Theis, M., Paton, A.W., Paton, J.C., Zimmermann, R., High, S.
(See online at https://doi.org/10.1371/journal.pone.0075394)
