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.
Highlights
Integral membrane proteins (IMPs) are central to cellular functions that include signal transduction, transport across the cell membrane, and energy conversion
Correlation between the topologies determined from simulations (x-axis) and compared to the experiments of Seppalaet al. (2010) (y-axis), reporting the fraction of fully integrated IMP configurations in the Ncyto/Ccyto topology
We utilize a recently developed CG computational approach (Zhang and Miller, 2012a) that enables the direct simulation of Sec-facilitated membrane integration of proteins on biological timescales to investigate the topogenesis of the dual-topology EmrE protein and its mutants
Summary
Integral membrane proteins (IMPs) are central to cellular functions that include signal transduction, transport across the cell membrane, and energy conversion. Performing these roles requires integration of the IMPs into the membrane with the correct topology (i.e., the correct orientation of the fully integrated IMP relative to the membrane). Membrane integration proceeds via the Sec translocon, a conserved protein-conducting channel located in the endoplasmic reticulum membrane in eukaryotes or in the plasma membrane in bacteria (White and von Heijne, 2004). The extent to which these factors influence the topology of multispanning IMPs is less clear
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