Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock

Abstract

Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes by an osmotic shock. They subsequently reseal into larger vesicles which, if necessary, can endure the same to obtain even bigger proteo-GUVs. This process does not require specific lipids or reagents, works under physiological conditions with high concentrations of protein, the proteins remains functional after incorporation and the resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is valid for a wide range of protein substrates. We have successfully reconstituted three structurally different proteins, two trans-membrane proteins, TolC and the neuronal t-SNARE, and one lipid-anchored peripheral protein, GABARAP-Like 1 (GL1). In each case, we verified that the protein remains active after incorporation and in their correctly folded state. We also measured their mobility by performing diffusion measurements via fluorescence recovery after photobleaching (FRAP) experiments on micromanipulated single GUVs. The diffusion coefficients are in agreement with previous data.

GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

“…2 uL of GABARAPL1-PE containing LUVs were deposited on 35-mm dish (MatTek), dried under atmospheric pressure, rehydrated with 6 uL water, dried again, and hydrated a last time with 6 uL water to produce GABARAPL1-GUVs 33 . The temperature for the hydration step should be carefully controlled, since the saturated lipid DPPC has a transition temperature equals to 41°C, the hydration should be done at a temperature ≥ 41°C; we set the temperature at 41°C to limit protein instability.…”

“…In addition, the stark puncta that we observe from GUV-GUV separation resembles the kinds of protein aggregates that sometimes form in cis on membranes and could suggest a collapse to a stable low-affinity cis-oligomer. In our GUV system, Fluorescence Recovery After Photobleaching (FRAP) experiments established that GL1 is freely diffusible and unlikely to form large cis-structures 33 , however FRAP is a bulk measurement, and we could miss small oligomers like dimers or trimers if they are present as part of a mix of different oligomeric states. In order to determine the range of stable available GL1-PE oligomeric states that normally form on a single membrane, we next performed single-oligomer bleaching experiments using a Total Internal Reflection Fluorescence (TIRF) microscope of Alexa488-GL1-PE incorporated into lipid bilayers formed directly on glass coverslip supports (Sup.…”

“…In vitro enzymatic lipidation using only ATG3 and ATG7 is highly sensitive to membrane curvature or to the presence of local membrane instabilities created by very large surface densities of conical lipids 28 , both properties that are inconsistent with GUV formation. Thus to facilitate GL1-PE integration into the essentially flat surface of a GUV, we recently described a novel GUV insertion protocol that relies upon first anchoring proteins in small liposomes and then fusing those vesicles via the osmotic-shock method 33 . GL1-PE reconstituted on to GUVs with this approach remains highly mobile, and diffuses with fast kinetics similar to individual lipids 33 , suggesting that Alexa488-GL1-PE exists as a monomer or potentially very small oligomer on GUVs.…”

“…Thus to facilitate GL1-PE integration into the essentially flat surface of a GUV, we recently described a novel GUV insertion protocol that relies upon first anchoring proteins in small liposomes and then fusing those vesicles via the osmotic-shock method 33 . GL1-PE reconstituted on to GUVs with this approach remains highly mobile, and diffuses with fast kinetics similar to individual lipids 33 , suggesting that Alexa488-GL1-PE exists as a monomer or potentially very small oligomer on GUVs.…”

“…included in lipidation reactions with Alexa488-GL1 (Green) either without ATP (producing proteinfree liposomes) or with ATP (to produce Alexa488-GL1-PE). These liposomes were subjected to the osmotic shock protocol 33 . In brief, liposomes were dried and resuspended in water two times to form GUVs.…”