13Helical membrane proteins constitute roughly a quarter of all proteomes and perform 14 diverse biological functions. To avoid aggregation, they undergo cotranslational membrane 15 insertion and are typically assumed to attain stable transmembrane topologies immediately 16 upon insertion. To what extent post-translational changes in topology are possible in-vivo and 17 how they may affect biogenesis is incompletely understood. Here, we show that monomeric 18 forms of Small Multidrug Resistance (SMR) proteins display topological dynamics, where the N-19 terminal transmembrane helix equilibrates between membrane-inserted and non-inserted 20 states. We characterize the kinetics of the process and show how the composition of the helix 21 regulates the topological dynamics. We further show that topological dynamics is a property of 22 the unassembled monomeric protein, as the N-terminal helix becomes fixed in a 23 transmembrane disposition upon dimerization. Membrane protein topology can thus remain 24 dynamic long after cotranslational membrane insertion, and can be regulated by later assembly 25 processes. 26 truncated or mutated to observe dynamics 9-13 . It remains unclear, however, if natural 48 membrane proteins can display topological dynamics under physiological conditions and, if so, 49 how such phenomena would affect the folding routes taken by membrane proteins in vivo. The 50 biophysical principles underlying topological dynamics are also poorly understood. 51Here we show that EmrE, a dimeric inner membrane protein from Escherichia coli, is 52 topologically dynamic in its pre-assembled monomeric state in vivo. We find that, in monomeric 53 EmrE, the N-terminal TMH1 flips in and out of the membrane, while dimeric EmrE has a stable 54 membrane topology. We characterize the kinetics of the process and show how changes in the 55 3 composition of TMH1 affect the rate of the topological dynamics. Our study suggests that 56 membrane proteins may exist in a dynamic equilibrium between different topological states, 57 and contributes to our understanding of how protein composition determines topology. 58 59
Results
60The N-terminus of monomeric EmrE is partially mislocalized 61 EmrE is an E. coli homodimeric multidrug transporter, composed of two 4-TMH 62 monomers 14 . It belongs to a unique group of proteins characterized by dual topology, where 63 the monomer inserts into the membrane with two opposite orientations with equal 64probabilities [15][16][17][18] . The oppositely-oriented monomers then assemble to form the functional 65 the N-terminus in the cytosol, with the Cys protected from blocking over a long period of time 132 (Fig. 3b). The slight blocking seen at long incubation times is similar to the background levels of 133 negative controls ( Supplementary Fig. 4b, Supplementary Fig. 5). 134We next asked if this phenomenon, of a monomer being topologically dynamic, is a rare 135 curiosity, or if it could perhaps be more common than expected. To examine this, we turned to 136 two distant homologs of EmrE ...