Cellular membranes are densely crowded with a diverse population of integral and membrane-associated proteins. In this complex environment, lipid rafts, which are phase-separated membrane domains enriched in cholesterol and saturated lipids, are thought to organize the membrane surface. Specifically, rafts may help to concentrate proteins and lipids locally, enabling cellular processes such as assembly of caveolae, budding of enveloped viruses, and sorting of lipids and proteins in the Golgi. However, the ability of rafts to concentrate protein species has not been quantified experimentally. Here we show that when membrane-bound proteins become densely crowded within liquid-ordered membrane regions, steric pressure arising from collisions between proteins can destabilize lipid phase separations, resulting in a homogeneous distribution of proteins and lipids over the membrane surface. Using a reconstituted system of lipid vesicles and recombinant proteins, we demonstrate that protein-protein steric pressure creates an energetic barrier to the stability of phase-separated membrane domains that increases in significance as the molecular weight of the proteins increases. Comparison with a simple analytical model reveals that domains are destabilized when the steric pressure exceeds the approximate enthalpy of membrane mixing. These results suggest that a subtle balance of free energies governs the stability of phase-separated cellular membranes, providing a new perspective on the role of lipid rafts as concentrators of membrane proteins.

have a cholate backbone with polar groups extending from one face and a short alkyl chain extending from the opposite face. FA-solubilized Cx26 crystallized into the H32 space group with two monomers in the asymmetric unit, and the crystals diffracted isotropically to 3.3-Å resolution. Using a molecular replacement search model based on a cryoEM map of Cx43 at 5.7-Å resolution [Fleishman et al., Mol. Cell 15: 879-888 (2004)], we solved the structure independently from a previously reported 3.5-Å resolution X-ray structure [2ZW3, Maeda et al., Nature 458: 597-602 (2009)]. The overall R/Rfree values and completeness were 0.311/0.328 and 98.9%, respectively, and the Molprobity score was 2.07 (100 th percentile). The RMS differences between 2ZW3 and our structure were 0.9 and 1.7-Å for the main-chain and side-chain atoms in the TM helices and 1.3 and 1.9-Å for the main-chain and side-chain atoms in E1 and E2. Although the topology and fold recapitulated 2ZW3, the maximum differences were significant: 2.7 and 5.9-Å for the main chain and side chain atoms in the TM helices and 3.9 and 7.6-Å for the main-chain and side-chain atoms in E1 and E2. We generated an electron density map at a resolution comparable to the cryoEM structure of the authentic Cx43 channel. The similarity of the maps suggests that detergent-solubilized Cx26 that crystallized as a dodecamer represents the authentic gap junction channel.