Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents

Abstract

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs.

Fig. 1.

Detergent-mediated reconstitution of transmembrane proteins in GUVs in physiological buffers. (A) Closed GUVs are grown by electroformation in the presence of subsolubilizing concentrations of mild detergents in sucrose solution (pink background). (B) The presence of detergent allows the fast equilibration of any physiological buffer (light purple background) between internal and external volumes and avoids the denaturation of solubilized transmembrane proteins. (C) Protein incorporation. (C, I) Purified and solubilized transmembrane proteins are unidirectionally reconstituted by direct insertion into GUVs. (C, II) Transmembrane proteins are reconstituted in GUVs after fusion of purified native vesicles in the presence of detergent. (D) Detergent is removed and GUVs are diluted in assay buffer that could be different from the internal buffer (salmon background).

Fig. 2.

Formation of GUVs in the presence of detergent and in physiological buffer. (A–D) Formation of EPC/EPA GUVs in the presence of detergent (DOTM) above the cmc. (A) No detergent. (B) Fifty micromolar DOTM, corresponding to the cmc. (C) One hundred micromolar DOTM. (D) Two hundred micromolar DOTM. (E–G) Exchange to buffers. GUVs formed in sucrose and 75 μM DOTM after dilution in a high-salt buffer without (E) or with (F) pyranine (green). DOTM GUVs are permeable to ions but not to large molecules (G) GUVs where DOTM was removed before buffer exchange, aggregate after dilution and in high-salt buffer.

Fig. 3.

Direct incorporation of solubilized transmembrane proteins in GUVs. Reconstitution of red-labeled bacteriorhodospin, a photoactivable proton pump, in DOTM destabilized EPC–EPA GUVs. (A and B) After detergent removal, light-induced proton pumping induces an internal acidification of GUVs. (C) Normalized pyranine fluorescence intensity and variance in the presence (black columns, n = 350) and in the absence (gray columns, n = 440) of valinomycin. (D) Schematic representation of the reconstitution of FhuA, a T5 phage receptor, in red-labeled EPC GUVs encapsulating YOPRO-1, a green fluorescent DNA probe. (E) After binding of T5 phage to FhuA, DNA is injected inside GUVs. (Scale bars: 10 μm.)

Fig. 4.

Incorporation of transmembrane proteins by fusion of native membranes with GUVs. Fusion of green-labeled inverted inner vesicles (IMVs) of E. coli containing overexpressed BmrC/BmrD, a multidrug resistance transporter to nonlabeled EPC–EPA GUVs in the absence (A) or the presence (B) of 300 μM Triton X-100. (C) Fusion of green-labeled chromatophores to DOPC/DOPE GUVs containing Tx-red–labeled lipid. (D) Fusion of proteoliposomes containing red-labeled bacteriorhodospin to unlabeled DOPC/DOPE GUVs in the presence of 300 μM Triton X-100. (E and F) Fusion of green E. coli IMVs to red-labeled GUVs containing lipid domains of DOPC/sphingomyelin/cholesterol, in the absence (E) or in the presence (F) of 300 μM Triton X-100. (Scale bars: 10 μm.)

Fig. 5.

Drug transport by BmrC/BmrD, a bacterial ABC transporter in GUVs. EPC/EPA GUVs are grown encapsulating DNA before incorporation of BmrC/BmrD by fusion of green E. coli IMVs. Addition of ATP induces the translocation of ethidium bromide (EtBr) and its binding to DNA induces an enhancement of its fluorescence. The transport is inhibited by addition of orthovanadate (Vi), a specific inhibitor of BmrC/BmrD. (Scale bars: 10 μm.)