Regulation of multispanning membrane protein topology via post-translational annealing

Abstract

The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

DOI: http://dx.doi.org/10.7554/eLife.08697.001

eLife digest

Proteins are long chains of smaller molecules called amino acids, and are built inside cells by a molecular machine called the ribosome. Many important proteins must be inserted into the membrane that surrounds each cell in order to carry out their role. As these proteins are being built by the ribosome, they thread their way into a membrane-spanning channel (called the translocon) from the inner side of the membrane. Short segments of these integral membrane proteins (called transmembrane domains) then become embedded in the membrane, while other parts of the protein remain on either side of the membrane.

For a membrane protein to work properly, the end of each of its transmembrane domains must be on the correct side of the membrane (i.e., the protein must obtain the correct ‘topology’). The conventional model for this process suggests that topology is fixed when the first transmembrane domain of a protein is initially integrated into the membrane, while the ribosome is still building the protein. This model can explain most integral membrane proteins, which only have a single topology. However, it cannot explain the family of membrane proteins that have an almost equal chance of adopting one of two different topologies (so-called ‘dual-topology proteins’).

Van Lehn et al. have now used computer modeling to simulate how a bacterial protein called EmrE (which is a dual-topology protein) integrates into the membrane via the translocon. The results reveal that a few transmembrane domains in EmrE do not fully integrate into the membrane while the ribosome is building the protein. Instead, these transmembrane domains slowly integrate after the ribosome has finished its job.

These findings contradict the conventional model and suggest that some membrane proteins only become fully integrated after the protein-building process is complete. The next step in this work is to experimentally test predictions from the computer simulations.

DOI: http://dx.doi.org/10.7554/eLife.08697.002