Probing Membrane Protein Sequestration and Oligomerization in Polymer-Tethered Phospholipid Bilayers Containing Raft-Mimicking Lipid Mixtures

Abstract

Lipid heterogeneities in plasma membranes are increasingly recognized to influence membrane protein distribution and function. Previous advancements in the understanding of such lipid-mediated protein sequestration processes have been rather sluggish, due to the small size and transient nature of lipid heterogeneities in cellular membranes. Therefore, theoretical approaches and model membrane studies have become important research tools. Recently, we introduced a powerful model membrane platform that allows the parallel analysis of membrane protein sequestering and oligomerization in well-defined lipid environments using confocal fluorescence intensity analysis paired with the photon counting histogram method. A hallmark of this single molecule-sensitive experimental strategy is the ability to monitor membrane proteins in micron-size membrane domains in the absence of artificial crosslinking agents. Specifically, we could show that native ligands, bilayer asymmetry, and cholesterol content have a profound effect on the sequestration of integrins in raft-mimicking lipid mixtures.1,2 These experimental findings can be explained on the basis of hydrophobic matching arguments and domain-specific lipid packing conditions. The described model membrane approach is also applied to investigate the oligomerization and sequestration behavior of GPI-anchored urokinase plasminogen activator receptor (uPAR). Here native ligands, but not cholesterol content, were found to impact dimerization level and sequestration of uPAR in raft-mimicking lipid mixtures.3 Corresponding experiments on mixtures of integrins and uPAR also illustrate the formation of integrin-uPAR complexes with distinct sequestration properties. Notably, these findings support a mechanism, in which GPI-anchored proteins cause the translocation of transmembrane proteins to lipid rafts.1) Siegel, A. P. et al. (2011) Biophys. J. 101, 1642.2) Hussain, N. F. et al. (2013) Biophys. J. 104, 2212.3) Ge, Y. et al. (2014) Biophys. J. (in press).