Functional ER architecture
Structural Biology
Cell Biology
Final Report Abstract
In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. Protein synthesis, transport and co-translational chemical modification are coupled at the mammalian endoplasmic reticulum (ER) by complex formation between the ribosome and the ER translocon. Structural dissection of this large complex, which is the beginning of the 'secretory pathway', is highly important because this pathway is absolutely essential for the cell in health and affected by diseases. For example, the human adaptive immune system relies on the secretory pathway and viruses target this pathway for their persistence and reproduction. The mammalian ER translocon consists of constitutive modules such as the Sec61 proteinconducting channel and the translocon associated protein complex (TRAP), as well as transient components such as the oligosaccharyltransferase (OST). TRAP regulates signal peptide and transmembrane helix insertion in a substrate-dependent manner, whereas OST is responsible for N-glycosylation of nascent proteins. Here, we used comparative cryo-electron tomography (ET) to dissect structure and function of TRAP and OST. We compared the mammalian TRAP structure with that in disease-linked TRAP mutant fibroblasts from human patients and with that in plants. The structural differences detected by subtomogram analysis form a basis for dissecting the molecular organization of the TRAP complex. We assigned positions to the four TRAP subunits within the complex, providing insights into their individual functions. Animals have two distinct types of OST complexes, which primarily differ in their catalytic subunit STT3a and STT3b, respectively. Using cryo-ET of STT3 knockouts we show that solely the STT3a-containing OST is part of the ribosome-translocon complex. Higher resolution tomographic analysis of the ribosome-Sec61-OST complex revealed how STT3A is integrated into the OST and how STT3 paralog specificity for translocon-associated OST is achieved. The revealed molecular architecture of the ER translocon advances our understanding of membrane protein biogenesis at the ER and sheds light on the role of TRAP in human congenital disorders of glycosylation.
Publications
- (2015). "Structure of the native Sec61 proteinconducting channel." Nat Commun 6: 8403
Pfeffer, S., L. Burbaum, P. Unverdorben, M. Pech, Y. Chen, R. Zimmermann, R. Beckmann and F. Förster
(See online at https://doi.org/10.1038/ncomms9403) - (2016). "Organization of the native ribosome-translocon complex at the mammalian endoplasmic reticulum membrane." Biochim Biophys Acta 1860(10): 2122-2129
Pfeffer, S., J. Dudek, R. Zimmermann and F. Förster
(See online at https://doi.org/10.1016/j.bbagen.2016.06.024) - (2016). "Visualizing the molecular sociology at the HeLa cell nuclear periphery." Science 351 (6276): 969-972
Mahamid, J., S. Pfeffer, M. Schaffer, E. Villa, R. Danev, L. K. Cuellar, F. Förster, A. A. Hyman, J. M. Plitzko and W. Baumeister
(See online at https://doi.org/10.1126/science.aad8857) - (2017). "Dissecting the molecular organization of the translocon-associated protein complex." Nat Commun 8: 14516
Pfeffer, S., J. Dudek, M. Schaffer, B. G. Ng, S. Albert, J. M. Plitzko, W. Baumeister, R. Zimmermann, H. H. Freeze, B. D. Engel and F. Förster
(See online at https://doi.org/10.1038/ncomms14516) - (2017). "Subtomogram analysis using the Volta phase plate." J Struct Biol 197(2): 94-101
Khoshouei, M., S. Pfeffer, W. Baumeister, F. Förster and R. Danev
(See online at https://doi.org/10.1016/j.jsb.2016.05.009) - (2018). "Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum." Science 360 (6385): 215-219
Braunger, K., S. Pfeffer, S. Shrimal, R. Gilmore, O. Berninghausen, E. C. Mandon, T. Becker, F. Förster and R. Beckmann
(See online at https://doi.org/10.1126/science.aar7899) - (2018). Structural Biology in Situ Using Cryo-Electron Subtomogram Analysis. Cellular Imaging: Electron Tomography and Related Techniques. E. Hanssen. Cham, Springer International Publishing: 237-259
Pfeffer, S. and F. Förster
(See online at https://doi.org/10.1007/978-3-319-68997-5_9)
