Determinants of Multispanning Membrane Protein Integration Mediated by the Sec Translocon

Multispanning membrane proteins - i.e., proteins with multiple transmembrane domains that thread back-and-forth across the cell membrane - are essential for cellular functions that include signal transduction, material transport, and energy conversion. Performing these functions requires the proteins to integrate within the membrane in the correct topology, or the overall orientation of the protein relative to the membrane. The integration of most proteins proceeds via the Sec translocon, a conserved protein-conducting channel that allows a nascent protein chain to access the membrane while being fed into the channel during translation. Previous studies have established that the Sec-facilitated integration of single-spanning proteins depends on factors including nascent chain hydrophobicity and charge; however, the extent to which these properties influence the integration and topology of multispanning proteins is less clear. Here, we use coarse-grained simulations to investigate the Sec-facilitated membrane integration of multispanning proteins on realistic biological timescales. We employ a simulation model that enables access to a timescale of minutes while retaining sufficient chemical accuracy to capture the forces that drive membrane integration. This study focuses on understanding two experimental observations: first, the finding that the protein EmrE exhibits two possible topologies in a 1:1 stoichiometry; and second, the finding that some marginally hydrophobic domains efficiently integrate into the membrane as part of a multispanning protein, despite inefficiently integrating as a single-spanning protein. This work provides mechanistic explanations for these observations, leading to insight into the sequence-level determinants of multispanning membrane protein integration and topology (1)