Membrane Protein Extraction by Styrene-Maleic Acid Copolymers is Independent of Subcellular Localization but is Influenced by Membrane Organization

Abstract

Styrene-Maleic acid (SMA) copolymers have emerged as a powerful alternative to detergents for applications in membrane research. Most notably, these amphipathic polymers can be used to directly extract and purify membrane proteins from intact cells of different organisms in the form of ‘‘native nanodiscs’’. These particles stabilize the protein in a near native environment comprising conserved native lipids as well as other membrane components that allow for the study of preferential interactions by biochemical analysis.To evaluate the general applicability of SMA-mediated membrane protein extraction from biomembranes, we employed fluorescence microscopy to visualize SMA solubilization of HeLa cells in real time. Our results indicate that SMA solubilization of (human) cells is a multi-step process that is not specific for any (sub)cellular membrane. However, we found solubilization kinetics of plasma membrane-localized proteins to vary markedly for different proteins, suggesting a strong influence of membrane organization and domain formation.To assess the influence of membrane properties on solubilization preferences of SMA in more detail, we employed an approach of partial solubilization of model membranes of different compositions. In lipid mixtures that form homogeneous bilayers, we found that SMA exhibits a high degree of promiscuity with respect to lipid headgroups. However, in phase-separating systems of fluid phases with either gel-phase or liquid-ordered phases, lipids in the fluid phase were solubilized with a strong preference.Our findings support the use of SMA for the study of lipid-protein interactions of membrane proteins and suggest applications for the isolation of membrane proteins from ordered domains in phase-separated membranes.