Membrane-Protein Diffusion in E. coli: A Random Walk in a Heterogeneous Landscape

Abstract

Membrane proteins perform vital cellular functions like respiration, signaling and nutrient uptake. For proper membrane-protein functioning, conformational dynamics, complex formation and ability to diffuse in the membrane are vital parameters. Despite a lot of work on model membranes, little is known about lateral diffusion of proteins in prokaryotic membranes. Here we use single-molecule wide-field epi-fluorescence microscopy to track, in living E. coli, the lateral mobility of seven trans-membrane proteins of different size fused to green fluorescent protein. We apply a novel method, IPODD (inverse projection of displacement distributions), to extract accurate diffusion coefficients from the 2-D projected diffusion trajectories along the 3-D curved bacterial membrane. The diffusion coefficients we find are significantly lower than those reported in in vitro studies of isolated membrane proteins in giant unilaminar vesicles. Our results indicate that crowding in the E. coli inner membrane substantially slows down trans-membrane protein mobility. Strikingly, we observe heterogeneity in the diffusive motion of all seven proteins: they all show a faster and a slower moving component. This heterogeneity does not appear to be connected to specific localization of the proteins in poles or other parts of the bacterium. Instead, our experiments indicate that the plasma membrane of E. coli contains patches with a different lipid composition than the bulk of the membrane, resulting in regions of slower and faster membrane-protein diffusion. These results show that the diffusion behavior of proteins embedded in the plasma membrane in E. coli is richer and far more complex than anticipated.