Formation of Giant Unilamellar Vesicles Containing Active Proteins

Abstract

Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model for cell membranes. However the study of proteo-membrane interactions in this system is limited because the incorporation of integral and lipid-anchored proteins into the GUVs remains challenging. All pre-existing protocols either produce membranes with a very low protein density or are tailored to specifically support the inclusion of a particular protein. We recently developed a simple generic method to incorporate protein in GUVs. It does not require specific lipids or reagents, works in physiological conditions with high concentrations of protein, and the resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is not limited to a narrow range of protein substrates; indeed we have already successfully incorporated two trans-membrane proteins and one lipid-anchored peripheral protein.These first proof-of-principle proteins present different types of challenges and thus demonstrate the broad utility of our method. TolC is an integral membrane protein and part of a heterotrimer, that together comprise a major multidrug efflux pump in E coli. The neuronal t-SNARE is a protein complex with a single transmembrane domain that mediates membrane fusion. Because of its propensity to aggregate t-SNARE is usually not functional after insertion in GUVs. To study lipidated proteins, we incorporated a modified form of the autophagy protein GABARAP L1, which we anchored it to the membrane via a cysteine-maleimide covalent bond. In each case, we verified that the proteins remain active after incorporation. We also verified their mobility by performing diffusion measurements via fluorescence recovery after photo bleaching (FRAP) experiments on micromanipulated GUVs. The diffusion coefficients are in agreement with previous data.