Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes

Abstract

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

Figure 1.

Bicelle-dilution lipid transfer measurement by lipid transfer proteins using fluorescence resonance energy transfer (FRET). (A) Structures of FRET energy donors (Me₄-BODIPY-GalCer, Me₄^-BODIPY-C1P) that are transferred by GLTP and CPTP, respectively, and nontransferable FRET energy acceptor (C18-DiI). (B) Excitation and emission spectra of Me₄-BODIPY-SL and C18-DiI. (C) FRET emission changes observed upon mixing POPC/DHPC donor bicelle-dilution model membranes containing Me₄-BODIPY-GalCer and C18-DiI with excess acceptor POPC/DHPC model membranes followed by GLTP. (D) Schematic showing GLTP-mediated transfer of Me₄-BODIPY-GalCer (lime green) out of POPC/DHPC bicelle-dilution “donor” vesicles containing nontransferable C18-DiI (red) to bicelle-dilution POPC/DHPC “acceptor” vesicles. (Bicelles and vesicles are not drawn to scale).

Figure 2.

Bicelle-dilution LTP assay: optimal performance conditions. (A) FRET changes reflect BODIPY-lipid transfer between donor and acceptor model membranes, not BODIPY-lipid binding by lipid transfer protein (GLTP). For black trace, a = donors added, b = acceptors added, and c = GLTP added. For red trace, a = donors added, b = GLTP added, and c = acceptors added. Response signals are nearly identical. (B) Effect of different q-value donors on GLTP transfer activity when mixed with 0.5 q-value acceptors. (C) Effect of different q-value acceptors on GLTP transfer activity when mixed with 0.5 q-value donors. a = donors added, b= acceptors added, and c = GLTP added. (D) Cryo-EM of 0.5 q-value dilution POPC/DHPC donors with FRET fluorphore lipids showing unilamellar nature and 31.5 ± 3.8 nm outer diameter. Bar graph shows vesicle size distribution. (E) Cryo-EM of 0.5 q-value dilution POPC/DHPC acceptors showing unilamellar nature and 36.3 ± 6.1 nm outer diameter. Bar graph shows vesicle size distribution.

Figure 3.

Bicelle-dilution LTP assay robustness. (A) GLTP accessibility to BODIPY-GalCer indicates bd-vesicles are stable and unilamellar. (B) Lack of dilution-induced structure changes to POPC/DHPC model membranes during the assay time course. 0.5 q-value donors (a) were mixed with 0.5 q-value acceptors (b) and equilibrated for various time intervals prior to GLTP addition (c). (C) Effects of different combinations of bd-vesicles versus conventional SUV donors and acceptors on GLTP transfer activity. Vesicles (s) = sonicated small vesicles; vesicles (e) = ethanol-injection small vesicles; donor and acceptor q-value mix = 0.5. (D) Superior stability of bd-vesicles improves FRET lipid transfer assay performance compared to conventional vesicles. Donors prepared in different ways were used in the transfer assay either soon after preparation or subjected to three freeze–thaw cycles (20 to −20 °C) prior to use. a = donors added (bd-vesicles or ethanol-injection vesicles), b = acceptors added (bd-vesicles or sonicated vesicles), and c = GLTP added.

Figure 4.

Applications of the bicelle-dilution LTP assay system. (A) Detection of Me₄-BODIPY-GalCer transfer (blue trace) by GLTP but not by ACD11 (red trace). (B) Detection of Me₄-BODIPY-C1P transfer by ACD11 (red trace) but not by GLTP (blue trace). q-Values = 0.5 for both donors and acceptors; a = donor addition, b = acceptor addition, and c = GLTP or ACD11 addition. (C) Nonfluorescent diethyl-C1P fails to slow BODIPY-C1P transfer, indicating no competition with BODIPY-C1P for interaction with ACD11 (i.e., no inhibition effect). (D) Slowing of ACD11 transfer of BODIPY-C1P by increasing levels of nonfluorescent C1P (1, 2, or 4 mol %) in bd-donor vesicles is indicative of competition for interaction with ACD11. (Donor q = 0.5; acceptor q = 0.5.) (E,F) In vivo transfer activity detection using the bd-assay by GLTP and CPTP expressed in HeLa cells after transient transfection. (E) GFP-GLTP or (F) GFP-CPTP: after transfection and growth for 24 h, the HeLa cells were disrupted by brief probe sonication (30 s × 2) on ice. Cell supernatants were prepared and used as described in the Methods section. (Donor q = 0.5; acceptor q = 0.5.)