Mechanistische Untersuchung der Assemblierung von Membranproteinkomplexen.
Zellbiologie
Zusammenfassung der Projektergebnisse
During the course of my work on the proposed project on the assembly of the model membrane protein complex EMC, an essential membrane protein biogenesis factor at the ER, my collaborators and I seized the exciting opportunity to solve its first three-dimensional structure. Our 3.4 Å cryo-EMC structure of the EMC revealed first insights into its highly conserved function as a transmembrane domain insertase. Using cell-based fluorescent reporter assays, we probed the effect of point mutations in individual EMC subunits on the insertion of its tail-anchored and multi-pass membrane protein substrates. We found that the EMC uses local membrane thinning and polar intramembrane residues in the subunits EMC3 and EMC6 to reduce the energetic barrier for insertion into the lipid bilayer. Our findings revealed novel insights into non-canonical membrane protein biogenesis by the EMC that are likely transferable to related insertases in all kingdoms of life. The structure of the EMC also allowed unprecedented insights into how such an intricate molecular machine might be assembled in the cell, which was the originally planned research objective of this proposal. Indeed, we found that assembly of the 9-subunit EMC from both soluble and membrane-spanning subunits is highly regulated and requires at least one assembly factor – the moonlighting kinase WNK1. We were able to show that WNK1 uses a conserved C-terminal amphipathic α-helix to protect the unassembled soluble subunit EMC2 from nonspecific interactions and premature degradation in the cell. WNK1 thus keep EMC2 in an assembly-competent state until it can be displaced by its soluble partner subunit EMC8. The EMC2-8 heterodimer is then capable of interacting with its membrane-bound interaction partners to assemble intact EMCs. Our work described a pipeline for the discovery and characterization of assembly factors and revealed general principles of how these factors safeguard the human proteome from toxic off-pathway interactions.
Projektbezogene Publikationen (Auswahl)
- (2020) Structural basis for membrane insertion by the human ER membrane protein complex. Science 369(6502):433-436
Pleiner, T.; Pinton Tomaleri, G.; Januszyk, K.; Inglis, A.J.; Hazu, M.; Voorhees, R.M.
(Siehe online unter https://doi.org/10.1126/science.abb5008) - (2021) WNK1 is an assembly factor for the human ER membrane protein complex. Mol. Cell, 81, 2693-2704.e12
Pleiner, T.; Hazu, M.; Pinton Tomaleri, G.; Januszyk, K.; Oania, R.S.; Sweredoski, M.J.; Moradian, A.; Guna, A.; Voorhees, R.M.
(Siehe online unter https://doi.org/10.1016/j.molcel.2021.04.013)
