Incorporation of Integral Membrane Proteins in Giant Lipid Vesicles by Swelling from a Protein-Loaded Hydrogel

Abstract

While giant unilamellar lipid vesicles (GUVs) have proven to be useful tools for probing the properties of biomembranes, the incorporation of integral membrane proteins into GUVs of controlled composition has remained challenging. We have developed a simple method for incorporating integral membrane proteins into the GUV membrane. Detergent-solubilized proteins are dissolved in an agarose hydrogel, which is spread as a thin film on a solid substrate. Lipids of the desired membrane composition are then deposited on top of the partially dehydrated, protein-containing hydrogel. The entire system is swollen with buffer following previously published protocols for forming GUVs from hydrogel-supported lipid films. The final detergent concentration in the rehydrated system is significantly lower than the critical micellar concentration of the detergent used to solubilize the membrane protein, which is transferred to the lipid bilayer of the GUV. GUVs form reliably and with good yield at a wide range of buffer ionic strengths and lipid compositions. High-molecular weight species can be encapsulated within the GUV interiors by including them in the rehydration buffer. We have used this system to demonstrate the cholesterol-dependent phase segregation of the alpha-helical transmembrane proteins aquaporin SoPIP2;1, aquaporin-Z, and bacteriorhodopsin. In lipid systems expected to undergo liquid ordered-liquid disordered phase segregation, these three proteins all consistently co-segregate with the cholesterol-poor phase as cholesterol concentration is varied from 20 to 50 mol%. We have also incorporated the human glucose transporter GLUT1 into GUV membranes and demonstrated its function. GUVs were fabricated encapsulating an enzymatic glucose assay with a fluorescent output. These GUVs were then immobilized in a microfluidic channel and a buffer containing glucose was introduced. Fluorescence was observed only in those GUVs fabricated with GLUT1 present in the agarose substrate.