SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2133172971> ?p ?o ?g. }

Showing items 1 to 100 of ±118 with 100 items per page.

W2133172971 endingPage "15568" @default.
W2133172971 startingPage "15559" @default.
W2133172971 abstract "Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop. Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop. In lipid bilayers the spontaneous movement of major phospholipids, e.g. of phosphatidylcholine (PC), 6The abbreviations used are: PC, phosphatidylcholine; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; ER, endoplasmic reticulum; GUV, giant unilamellar vesicles; LPC, l-α-lysophosphatidylcholine; PE, phosphatidylethanolamine; TE, Triton X-100 extract; RT, room temperature; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; C6-acyl-PC, 1-acyl-2-hexanoyl-sn-glycero-3-PC; C6-NBD-PC, 1-palmitoyl-2-(NBD-hexanoyl)-sn-glycero-3-PC.6The abbreviations used are: PC, phosphatidylcholine; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; ER, endoplasmic reticulum; GUV, giant unilamellar vesicles; LPC, l-α-lysophosphatidylcholine; PE, phosphatidylethanolamine; TE, Triton X-100 extract; RT, room temperature; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; C6-acyl-PC, 1-acyl-2-hexanoyl-sn-glycero-3-PC; C6-NBD-PC, 1-palmitoyl-2-(NBD-hexanoyl)-sn-glycero-3-PC. between the two monolayers is slow, with half-times on the order of hours or even days. However, lipid topology of cellular membranes results from a continuous bidirectional movement (flip-flop) of lipids between the two leaflets in which specific membrane proteins, so called flippases, play an essential role (1Kornberg R.D. McConnell H.M. Biochemistry. 1971; 10: 1111-1120Crossref PubMed Scopus (771) Google Scholar, 2Holthuis J.C.M. Levine T.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 209-220Crossref PubMed Scopus (408) Google Scholar). Energy-independent flippases allow phospholipids to equilibrate rapidly between the two monolayers, whereas energy-dependent flippases mediate a net transfer of specific phospholipids to one leaflet of the membrane. Candidates for the latter flippase are members of a conserved subfamily of P-type ATPases (2Holthuis J.C.M. Levine T.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 209-220Crossref PubMed Scopus (408) Google Scholar) as well as ATP binding cassette transporters (3Pohl A. Devaux P.F. Herrmann A. Biochim. Biophys. Acta. 2005; 1733: 29-52Crossref PubMed Scopus (124) Google Scholar).In eukaryotes the cytoplasmic leaflet of the endoplasmic reticulum (ER) membrane is the major site of phospholipid biosynthesis. To ensure stable membrane growth, energy-independent flippases mediate rapid, bidirectional, and rather unspecific phospholipid flip-flop with half-times of minutes or less (4Bishop W.R. Bell R.M. Cell. 1985; 42: 51-60Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 5Herrmann A. Zachowski A. Devaux P.F. Biochemistry. 1990; 29: 2023-2027Crossref PubMed Scopus (111) Google Scholar, 6Buton X. Morrot G. Fellmann P. Seigneuret M. J. Biol. Chem. 1996; 271: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7Marx U. Lassmann G. Holzhütter H.G. Wüstner D. Müller P. Höhlig A. Kubelt J. Herrmann A. Biophys. J. 2000; 78: 2628-2640Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). A similar flippase activity was also found in the bacterial inner membrane, where lipid synthesis occurs likewise at the cytoplasmic leaflet (8Hrafnsdóttir S. Nichols J.W. Menon A.K. Biochemistry. 1997; 36: 4969-4978Crossref PubMed Scopus (57) Google Scholar).Techniques to determine transbilayer phospholipid movement as well as the activity of flippases have been critically evaluated (9Devaux P.F. Fellmann P. Hervé P. Chem. Phys. Lipids. 2002; 116: 115-134Crossref PubMed Scopus (81) Google Scholar). Spin-labeled and fluorescent lipid analogues have provided much insight into protein-mediated transbilayer dynamics of phospholipids (9Devaux P.F. Fellmann P. Hervé P. Chem. Phys. Lipids. 2002; 116: 115-134Crossref PubMed Scopus (81) Google Scholar). However, the bulky reporter moieties may affect the absolute values of transbilayer lipid movement.An alternative approach to characterize flip-flop is based on shape changes of deflated, prolate giant unilamellar vesicles (GUVs) (10Mui B.L. Döbereiner H.G. Madden T.D. Cullis P.R. Biophys J. 1995; 69: 930-941Abstract Full Text PDF PubMed Scopus (109) Google Scholar), which do not require labeled lipid analogues. Shape transitions can be triggered by a very small excess of lipid in one monolayer (11López-Montero I. Rodriguez N. Cribier S. Pohl A. Velez M. Devaux P.F. J. Biol. Chem. 2005; 280: 25811-25819Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 12Berndl K. Käs J. Lipowsky R. Sackmann E. Seifert U. Europhys. Lett. 1990; 13: 659-664Crossref Scopus (194) Google Scholar, 13Farge E. Devaux P.F. Biophys. J. 1992; 61: 347-357Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 14Tanaka T. Sano R. Yamashita Y. Yamazaki M. Langmuir. 2004; 20: 9526-9534Crossref PubMed Scopus (84) Google Scholar). For example, unlabeled lipids were inserted into the external leaflet of GUVs made of egg-PC (11López-Montero I. Rodriguez N. Cribier S. Pohl A. Velez M. Devaux P.F. J. Biol. Chem. 2005; 280: 25811-25819Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Because of the low compressibility of lipid monolayers, the asymmetric lipid supply creates a surface area difference between the two leaflets, which in turn results in formation of a bud-like structure (Fig. 1A). If lipids (e.g. lyso-PC, LPC) that are known to undergo a very slow flip-flop similar to that of egg-PC were added, the bud was stable, whereas if the added lipids (e.g. ceramide) were able to rapidly redistribute between the two membrane leaflets, the initial bud formation was reversible, and the prolate shape was recovered (11López-Montero I. Rodriguez N. Cribier S. Pohl A. Velez M. Devaux P.F. J. Biol. Chem. 2005; 280: 25811-25819Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). This can be explained by a relaxation of the monolayer area difference due to the transbilayer equilibration of the inserted lipids. The time dependence of shape changes could be used for derivation of the flip-flop rate constant.Here we demonstrate by reconstitution of yeast ER flippase activity into GUVs that dynamics of GUV shape changes can serve as a tool for quantitative characterization of the lipid transport activity of flippases (Fig. 1B). In this case the characteristic time of the sequential shape transformation of protein-containing GUVs is on the order of flip-flop of spin-labeled and fluorescent phospholipid analogues found previously in microsomes (7Marx U. Lassmann G. Holzhütter H.G. Wüstner D. Müller P. Höhlig A. Kubelt J. Herrmann A. Biophys. J. 2000; 78: 2628-2640Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The major advantage of this approach compared with the previous ones is the use of unlabeled lipids, which allows avoiding possible artifacts related to the utilization of lipid analogues with bulky reporter moieties (9Devaux P.F. Fellmann P. Hervé P. Chem. Phys. Lipids. 2002; 116: 115-134Crossref PubMed Scopus (81) Google Scholar).EXPERIMENTAL PROCEDURESMaterials—Egg l-α-lysophosphatidylcholine (egg-LPC), egg phosphatidylcholine (egg-PC), egg phosphatidylethanolamine (egg-PE), and dioleoylphosphatidylglycerol (PG) were from Sigma-Aldrich. 1-Acyl-2-hexanoyl-sn-glycero-3-phoshatidylcholine (C6-acyl-PC) was synthesized from egg-PC (15Fellmann P. Zachowski A. Devaux P.F. Methods Mol. Biol. 1994; 27: 161-175PubMed Google Scholar). C6-ceramide, C16-ceramide, and galactosylceramide were synthesized according to López-Montero et al. (11López-Montero I. Rodriguez N. Cribier S. Pohl A. Velez M. Devaux P.F. J. Biol. Chem. 2005; 280: 25811-25819Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). 7-Diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), BODIPY FLC5-PC and -Cer were purchased from Molecular Probes (Eugene, OR).Yeast Strains and Culture Conditions—The yeast strain sec61 (MATα ura3-52 leu2-3-112 ade2-10 sec61ts) obtained from J. Holthuis (University Utrecht, The Netherlands) was used. Microsomal membranes containing a green fluorescent protein-tagged transmembrane domain linked to carboxypeptidase yscY (for details, see Ref. 16Taxis C. Hitt R. Park S.H. Deak P.M. Kostova Z. Wolf D.H. J. Biol. Chem. 2003; 278: 35903-35913Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) were derived from W303-1C Δder3 (MATα ade2-1 ura3-1 his3-11, 15 leu2-3, 112 trp1-1 can1-100 prc1-1, Δder3::HIS3) harboring the plasmid pMA1 (kindly provided by D. Wolf, University Stuttgart, Germany). Strains were grown at 27 °C in liquid YPD medium (1% Bacto yeast extract, 2% Bacto peptone (Difco, 2% glucose). The strain W303-1C Δder3 harboring pMA1 was grown in the absence of uracil in synthetic complete medium with 2% glucose at 27 °C. Cells were harvested in the exponential phase, and growth was monitored by A600.Microsome Preparation—Microsomes were prepared essentially according to Zinser and Daum (17Zinser E. Daum G. Yeast. 1995; 11: 493-536Crossref PubMed Scopus (301) Google Scholar). Briefly, cells were pretreated with 10 mm dithiothreitol in 100 mm Tris sulfate (pH 9.4) for 10 min, incubated with Zymolyase-100T (ICN Biomedicals, Eschwege, Germany) at 5 mg/g of cells (wet weight) in buffer (20 mm potassium phosphate (pH 7.2), 1.2 m sorbitol) at 30 °C for 30 min, harvested, and then lysed in homogenizing buffer (10 mm Tris-HCl (pH 7.4), 0.6 m sorbitol) containing protease inhibitors (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 5 mg/ml antipain, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride) using a Dounce homogenizer with a tight-fitting pestle. The cell lysate was centrifuged at 3000 × g for 5 min. The pellet was resuspended in homogenizing buffer, homogenized, and centrifuged as above. Both supernatants were pooled. Membrane pellets from differential centrifugation (9,000 × g for 10 min, 20,000 × g for 30 min, 40,000 × g for 30 min, 100,000 × g for 45-75 min) were screened for organelle marker proteins by immunoblotting. The green fluorescent protein-tagged ER membrane protein became enriched in the 40,000 × g fraction. Pellets of 40,000 × g and 100,000 × g enriched in ER membrane proteins were resuspended in buffer T (10 mm Tris-HCl (pH 7.4), 1 mm EDTA) using a tight fitting Dounce homogenizer and stored at -80 °C. If not stated otherwise, the 100,000 × g fraction was used for further preparation of proteoliposomes.Preparation of Proteoliposomes—Microsomal fractions (20 mg/ml protein) were solubilized by diluting half with 1.6% (w/v) Triton X-100 (Roche Diagnostics) in buffer T. After incubation for 45 min on ice, insoluble material was removed by centrifugation (177,000 × g, 30 min, 4 °C). The resulting supernatant is designated as Triton extract (TE). For reconstitution, egg-PC in chloroform was dried under nitrogen in a glass tube and then dissolved in buffer TX (10 mm Tris-HCl (pH 7.4), 1 mm EDTA, 0.8% w/v Triton X-100) to a final concentration of 4.5 mm. Various amounts of TE were added to generate proteoliposomes with different lipid/protein ratios. Protein-free liposomes were prepared similarly by substituting buffer for TE. To remove detergent and generate vesicles, 100 mg of SM2 BioBeads (Bio-Rad) per 1 ml of solution were added. After 3-4 h rotating at room temperature (RT), the samples were supplemented with an additional 200 mg/ml of BioBeads and transferred to 4 °C for further 14-16 h of mixing. Turbid suspensions were withdrawn, carefully avoiding collection of any beads, and adjusted to an 8-10-fold excess of buffer T. The vesicles were collected by centrifugation (200,000 × g, 50 min, 4 °C) and resuspended at 9 mm phospholipid using a Dounce homogenizer. Protein recovery in the reconstituted vesicles was about 30-45%; lipid recovery was about 90%. More than 99.9% of the original Triton X-100 was removed, as determined by extraction with four volumes of chloroform/methanol (1/2, v/v) and subsequent measurement of the supernatant at A275.To label microsomal proteins before reconstitution, 450 μl of TE (0.9 mg protein) were mixed with 20 μl of a 10 mm Me2SO solution of the thiol-reactive fluorescent probe CPM and incubated at 4 °C overnight in the dark. Unbound label was removed by dialysis. Analysis by SDS-PAGE and thin layer chromatography demonstrated that at this temperature only proteins but no membrane lipids became labeled (data not shown).Virosome Preparation—Virosomes were prepared from influenza viruses (strain X31) essentially as described for proteoliposomes. A mixture of virosomal TE, Triton X-100-solubilized egg-PC, and egg-PE (14.75/0.275, w/w) was used for reconstitution at lipid/protein ratio of 20 (w/w). To visualize reconstitution of viral proteins, viruses were preincubated for 2 h at RT in the dark with CPM, added from 5 mm Me2SO stock to molar excess of 10 over hemagglutinin. To remove uncoupled CPM, labeled viruses were washed twice in phosphate-buffered saline (pH 7.4) and harvested by centrifugation at 45,000 × g. SDS-PAGE analysis revealed that only influenza virus hemagglutinin was labeled (data not shown).Preparation of Giant Unilamellar Vesicles—GUVs were generated from proteoliposomes by the electroformation technique in a chamber made of indium-tin-oxide-coated glass slides at RT (18Angelova M. Soleau S. Meleard P. Faucon J.F. Bothorel P. Prog. Colloid. Polym. Sci. 1992; 89: 127-131Crossref Google Scholar, 19Mathivet L. Cribier S. Devaux P.F. Biophys. J. 1996; 70: 1112-1121Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 20Girard P. Pécréaux J. Lenoir G. Falson P. Rigaud J-L. Bassereau P. Biophys. J. 2004; 87: 419-429Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The oscillating electric field that was applied only during GUV formation did not affect the activity of membrane proteins (20Girard P. Pécréaux J. Lenoir G. Falson P. Rigaud J-L. Bassereau P. Biophys. J. 2004; 87: 419-429Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). For GUVs consisting only of lipids, 70 μl of lipid solution in chloroform (0.25 mg/ml) were deposited in small droplets onto each slide. Subsequently, solvent was evaporated in a desiccator (10 mbar) for 60 min at RT. To prepare GUVs with reconstituted proteins according to Girard et al. (20Girard P. Pécréaux J. Lenoir G. Falson P. Rigaud J-L. Bassereau P. Biophys. J. 2004; 87: 419-429Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), proteoliposomes were diluted with distilled water to 0.4 - 0.8 mg/ml lipid, and 25-50 μl of this suspension was deposited onto each glass slide in small droplets. To avoid denaturation, proteoliposomes were dehydrated only partially by placing glass slides in a sealed chamber containing a saturated NaCl solution. After overnight incubation a film had formed on the glass slides.Subsequently, the chamber was assembled by sealing both glass slides with silicon paste. The sucrose solution (250 mm sucrose, 0.02% NaN3) was injected with a syringe (about 1 ml) through a micropore filter immediately before connecting the completely sealed chamber to the generator. The voltage of the applied AC-field was increased stepwise every 6 min from 20 mV up to 1.1 V while continuously increasing the frequency from 4 to 10 Hz within the first minute. The AC field was applied for 3-12 h. To complete the procedure voltage was raised to 1.3 V, and the frequency was lowered to 4 Hz for 1 h. The chamber was then stored in a refrigerator at 4 °C.Observation and Analysis of GUV Shape Changes—Spherical GUVs cannot undergo shape changes due to the minimized surface to volume ratio. Therefore, deflated vesicles appropriate for shape change studies (11López-Montero I. Rodriguez N. Cribier S. Pohl A. Velez M. Devaux P.F. J. Biol. Chem. 2005; 280: 25811-25819Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) were achieved osmotically by transferring 10-50 μl of GUVs into 150 μl of a glucose solution (275 mm glucose, 0.02% Na3) on a microscope slide and allowing water to evaporate for 30-60 min at room temperature. To prolate GUVs, lipids that are known to incorporate rapidly into lipid bilayers were added. Typically, 10 μl of 0.1 mm lipid (typically egg-LPC) in glucose solution was injected (representing time zero in the shape changes analysis). The time points t1 (begin of budding transition) and t2 (begin of bud retraction) were measured by microscopic observation of GUV shapes for each experiment (see “Results”). All experiments were performed at room temperature. To determine the amount of lipid incorporated into GUVs, we used radioactive l-lyso-3-phosphatidylcholine,1-[1-14C]palmitoyl (CFA633, Amersham Biosciences). 50 μl of GUVs were transferred to a microscope slide, and 150 μl of a glucose solution (275 mm glucose, 0.02% NaN3) was added. After GUVs settled, radioactive lipids were added, and the suspension was incubated for up to 2 h. Subsequently, aliquots from the supernatant were taken, and radioactivity was measured.Miscellaneous—Protein content was quantified after TCA precipitation using micro-BCA Protein Assay reagent (Pierce) and BSA as a standard. Phospholipid content was determined by extracting lipids according to Bligh and Dyer (21Bligh E.G. Dyer W.J. Can. J. Med. Sci. 1959; 37: 911-917Google Scholar) and measuring the amount of phospholipid phosphorus (22Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2859) Google Scholar).RESULTSShape Changes of Protein-free GUVs—Insertion of unlabeled lipids such as egg-LPC, C6-acyl-PC, or C6-ceramide into the external leaflet of GUVs made of egg-PC resulted in formation of a bud-like structure (Figs. 1A and 2). If egg-LPC or C6-acyl-PC, which are known to undergo a very slow flip-flop like egg-PC, was added, the bud was stable (Fig. 2). Whereas if lipids were added that rapidly redistribute between the two membrane leaflets, e.g. ceramide, the initial bud was reversible, and the prolate shape was recovered (11López-Montero I. Rodriguez N. Cribier S. Pohl A. Velez M. Devaux P.F. J. Biol. Chem. 2005; 280: 25811-25819Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). This can be explained by a decline in the monolayer area difference due to the transbilayer equilibration of inserted lipids. Bud formation was also reversible when egg-LPC was added to egg-PC GUVs containing already a lipid that flips rapidly, such as the long chain C16-ceramide (5 mol %) (not shown). When ceramide was replaced by the slow flipping galactosylceramide, the prolate shape was not recovered after 1 h. The experiments with ceramide and egg-LPC prove that the transversal diffusion of an unlabeled lipid with a fast flip-flop rate can be measured in GUVs even disregarding the flipping ability of the added lipid. However, this conclusion may not be generalized. Although cholesterol is known to flip rapidly across the lipid bilayer (23Leventis R. Silvius J.R. Biophys. J. 2001; 81: 2257-2267Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 24John K. Kubelt J. Müller P. Wüstner D. Herrmann A. Biophys. J. 2002; 83: 1525-1534Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), Mathivet et al. (19Mathivet L. Cribier S. Devaux P.F. Biophys. J. 1996; 70: 1112-1121Abstract Full Text PDF PubMed Scopus (235) Google Scholar) did not observe retraction of the initial bud formation when egg-LPC was added to egg-PC GUVs containing up to 23 mol % of cholesterol. A possible explanation could be that transbilayer redistribution of cholesterol cannot compensate for area asymmetry due its specific molecular properties, e.g. it is known that cholesterol cannot form bilayers.FIGURE 2Shape transition of egg-PC GUVs upon the addition of egg-LPC. Insertion of egg-LPC into the outer leaflet caused the formation of a stable bud that did not retract in the time course of the experiment (for details, see “Experimental Procedures”).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To substantiate our conclusions on lipid translocation drawn from GUV shapes, we chose further processes exploiting microscopic visualization of GUV behavior. First, Cullis and co-workers (Ref. 25Hope M.J. Redelmeier T.E. Wong K.F. Rodrigueza W. Cullis P.R. Biochemistry. 1989; 28: 4181-4187Crossref PubMed Scopus (99) Google Scholar and publications cited therein) demonstrated that pH gradients influence the distribution of charged phospholipids as phosphatidylglycerol (PG) across the membrane of unilamellar 100-nm vesicles. Using various assays they could prove that PG flips rapidly in its neutral form, whereas it is sequestered within one membrane leaflet in the case of deprotonation in contact with basic environment. Thereby, arising transversal asymmetry could clearly be related to membrane curvature and molecule balance between the leaflets in GUVs (13Farge E. Devaux P.F. Biophys. J. 1992; 61: 347-357Abstract Full Text PDF PubMed Scopus (205) Google Scholar). When prolate GUVs from egg-PC containing 1 mol % of dioleoyl-PG are exposed to an increased pH (from pH 7 to 9) in the surrounding medium by the addition of NaOH, spontaneous migration of PG from the inner leaflet (pH 7) to the exterior (pH 9) results in relative immobilization due to their acquired head-group charge. The excess molecules account for an area imbalance that is apparent from an instantaneous budding transition (Fig. 3A). The connecting neck is progressively elongated during the following minutes, eventually forming a tether with a highly mobile daughter vesicle (see the change of focus to resolve both structures in last images of series). Control GUVs from egg-PC maintain their prolate shapes for more than 10 min (not shown). By a second independent approach we proved directly that shape changes indeed correlate with the transbilayer distribution of externally added lipids using BODIPY-labeled PC and Cer (see supplemental Fig. 1). Quantification of the fluorescent fraction on the inner surface of the membrane, as determined by protection from trypan blue quenching, is displayed in Fig. 3B. BODIPY-PC proves to be confined essentially to the outer leaflet, whereas the distribution of the Cer analogue resembles that of GUVs prepared with a symmetric BODIPY-PC distribution between both leaflets. We, therefore, conclude that lipid distribution affects the membrane curvature of GUVs, and its dynamics can be reliably visualized as shape changes.FIGURE 3Shape changes of GUVs correlate with transbilayer redistribution of lipids. A, shape changes with pH-induced flip-flop of phosphatidylglycerol. GUVs were prepared from egg-PC containing 1 mol % dioleoylphosphatidylglycerol. Shown is prolate GUV at pH 7 (a) subjected to pH increase (pH 9) by adding ∼10 μl of NaOH (0.5 n) to ∼400 μl of neutral glucose solution in the observation chamber (b). Phase contrast images are taken with respect to time of alkaline addition (0′00″); the bar corresponds to 10 μm. B, assessment of transbilayer distribution of BODIPY-PC and BODIPY-Cer after shape transitions. BODIPY-PC or -Cer were added to GUVs and incubated for 30 and 10 min, respectively. Subsequently, fluorescence intensities in the absence and presence of the quencher trypan blue (0.0075% w/v) were measured (as described in the supplemental data). Intensity in the absence of trypan blue was set to 1. For comparison, results for GUVs symmetrically labeled with BODIPY-PC are shown (BODIPY-PC*). Data shown are the means ± S.E. of four determinations. Note that fluorescence intensities in the presence of trypan blue do not correlate in a quantitative manner with transbilayer distribution of BODIPY-lipids. For that, higher concentrations of the quencher would be necessary. However, such concentrations affect the stability of the GUVs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Shape Changes of GUVs with Reconstituted ER Proteins—The flippase activity of yeast ER was first reconstituted into proteoliposomes as already described previously for rat liver (6Buton X. Morrot G. Fellmann P. Seigneuret M. J. Biol. Chem. 1996; 271: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 7Marx U. Lassmann G. Holzhütter H.G. Wüstner D. Müller P. Höhlig A. Kubelt J. Herrmann A. Biophys. J. 2000; 78: 2628-2640Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and yeast ER (26Nicolson T. Mayinger P. FEBS Lett. 2000; 476: 277-281Crossref PubMed Scopus (19) Google Scholar). Applying established assays for measuring transbilayer movement (see Ref. 9Devaux P.F. Fellmann P. Hervé P. Chem. Phys. Lipids. 2002; 116: 115-134Crossref PubMed Scopus (81) Google Scholar and references therein) confirmed protein-mediated flip-flop of fluorescent phospholipid analogues such as 1-palmitoyl-2-(NBD-hexanoyl)-sn-glycero-3-phosphocholine (C6-NBD-PC) being on the order of a minute. 7S. Vehring, A. Herrmann, and T. Pomorski, unpublished data. Subsequently, GUVs were prepared from such proteoliposomes. No significant difference between the size of protein-free and protein-containing GUVs was found (not shown).When egg-LPC or C6-acyl-PC was added, reconstituted GUVs budded within the first 5 min after injection of lipids and subsequently regained the prolate shape within 5-10 min (Fig. 4). We conclude that the reconstituted ER flippase activity triggers the rapid flip-flop that diminishes the surface area difference between the two leaflets. To demonstrate that GUVs undergoing reversible shape changes contain ER proteins, we reconstituted the ER fraction from a yeast strain expressing an ER resident, green fluorescent protein-tagged membrane protein as well as prelabeled TE (supplemental Figs. 3 and 4).FIGURE 4Shape transition of GUVs prepared from proteoliposomes containing microsomal membrane proteins (100,000 × g fraction). At time t = 0, egg-LPC was added. GUVs were visualized by differential interference contrast at RT. The lipid/protein ratio was 35/1 (w/w). The budding transition was observed at t1 = 3:25 min. Recovery of the prolate shape started at t2 = 10:35 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Shape Changes of GUVs Reconstituted with Influenza Virus Protein—To address whether the flippase activity is a property of specific membrane proteins (27Kol M.A. van Dalen A. de Kroon A.I. de Kruijff B. J. Biol. Chem. 2003; 278: 24586-24593Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), we studied shape changes of GUVs prepared from reconstituted influenza virosomes. Upon the addition of egg-LPC, GUVs formed stable buds (Fig. 5)." @default.
W2133172971 created "2016-06-24" @default.
W2133172971 creator A5014112590 @default.
W2133172971 creator A5018045578 @default.
W2133172971 creator A5021200899 @default.
W2133172971 creator A5027205756 @default.
W2133172971 creator A5032996487 @default.
W2133172971 creator A5033530736 @default.
W2133172971 creator A5040835021 @default.
W2133172971 creator A5062237124 @default.
W2133172971 creator A5062465955 @default.
W2133172971 date "2007-05-01" @default.
W2133172971 modified "2023-10-15" @default.
W2133172971 title "Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles" @default.
W2133172971 cites W1968273291 @default.
W2133172971 cites W1968476108 @default.
W2133172971 cites W1968968981 @default.
W2133172971 cites W1970123407 @default.
W2133172971 cites W1971641980 @default.
W2133172971 cites W1973655670 @default.
W2133172971 cites W1979112749 @default.
W2133172971 cites W1981020955 @default.
W2133172971 cites W1988857297 @default.
W2133172971 cites W2001834121 @default.
W2133172971 cites W2003709444 @default.
W2133172971 cites W2009110742 @default.
W2133172971 cites W2010918799 @default.
W2133172971 cites W2015636833 @default.
W2133172971 cites W2016267013 @default.
W2133172971 cites W2016641591 @default.
W2133172971 cites W2037385253 @default.
W2133172971 cites W2041991446 @default.
W2133172971 cites W2047361910 @default.
W2133172971 cites W2047910658 @default.
W2133172971 cites W2048658013 @default.
W2133172971 cites W2049656093 @default.
W2133172971 cites W2052653429 @default.
W2133172971 cites W2073371381 @default.
W2133172971 cites W2075530070 @default.
W2133172971 cites W2083180901 @default.
W2133172971 cites W2124378189 @default.
W2133172971 cites W2133529176 @default.
W2133172971 cites W2147918760 @default.
W2133172971 cites W2149026678 @default.
W2133172971 cites W2150405813 @default.
W2133172971 cites W2158261605 @default.
W2133172971 cites W2165068711 @default.
W2133172971 cites W2237164154 @default.
W2133172971 doi "https://doi.org/10.1074/jbc.m604740200" @default.
W2133172971 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17369612" @default.
W2133172971 hasPublicationYear "2007" @default.
W2133172971 type Work @default.
W2133172971 sameAs 2133172971 @default.
W2133172971 citedByCount "62" @default.
W2133172971 countsByYear W21331729712012 @default.
W2133172971 countsByYear W21331729712013 @default.
W2133172971 countsByYear W21331729712014 @default.
W2133172971 countsByYear W21331729712015 @default.
W2133172971 countsByYear W21331729712016 @default.
W2133172971 countsByYear W21331729712017 @default.
W2133172971 countsByYear W21331729712018 @default.
W2133172971 countsByYear W21331729712019 @default.
W2133172971 countsByYear W21331729712020 @default.
W2133172971 countsByYear W21331729712021 @default.
W2133172971 countsByYear W21331729712022 @default.
W2133172971 countsByYear W21331729712023 @default.
W2133172971 crossrefType "journal-article" @default.
W2133172971 hasAuthorship W2133172971A5014112590 @default.
W2133172971 hasAuthorship W2133172971A5018045578 @default.
W2133172971 hasAuthorship W2133172971A5021200899 @default.
W2133172971 hasAuthorship W2133172971A5027205756 @default.
W2133172971 hasAuthorship W2133172971A5032996487 @default.
W2133172971 hasAuthorship W2133172971A5033530736 @default.
W2133172971 hasAuthorship W2133172971A5040835021 @default.
W2133172971 hasAuthorship W2133172971A5062237124 @default.
W2133172971 hasAuthorship W2133172971A5062465955 @default.
W2133172971 hasBestOaLocation W21331729711 @default.
W2133172971 hasConcept C12554922 @default.
W2133172971 hasConcept C130316041 @default.
W2133172971 hasConcept C185592680 @default.
W2133172971 hasConcept C2777019660 @default.
W2133172971 hasConcept C2778918659 @default.
W2133172971 hasConcept C2779637612 @default.
W2133172971 hasConcept C3018040386 @default.
W2133172971 hasConcept C41625074 @default.
W2133172971 hasConcept C55493867 @default.
W2133172971 hasConcept C86803240 @default.
W2133172971 hasConceptScore W2133172971C12554922 @default.
W2133172971 hasConceptScore W2133172971C130316041 @default.
W2133172971 hasConceptScore W2133172971C185592680 @default.
W2133172971 hasConceptScore W2133172971C2777019660 @default.
W2133172971 hasConceptScore W2133172971C2778918659 @default.
W2133172971 hasConceptScore W2133172971C2779637612 @default.
W2133172971 hasConceptScore W2133172971C3018040386 @default.
W2133172971 hasConceptScore W2133172971C41625074 @default.
W2133172971 hasConceptScore W2133172971C55493867 @default.
W2133172971 hasConceptScore W2133172971C86803240 @default.
W2133172971 hasIssue "21" @default.