Abstract
Herein, we describe methodological approaches for measuring in vitro transfer of sphingolipids (SLs) between membranes. The approaches rely on direct tracking of the lipid. Typically, direct tracking involves lipid labeling via attachment of fluorophores or introduction of radioactivity. Members of the GlycoLipid Transfer Protein (GLTP) superfamily are used to illustrate two broadly applicable methods for direct lipid tracking. One method relies on Förster resonance energy transfer (FRET) that enables continuous assessment of fluorophore-labeled SL transfer in real time between lipid donor and acceptor vesicles. The second method relies on tracking of radiolabeled SL transfer by separation of lipid donor and acceptor vesicles at discrete time points. The assays are readily adjustable for assessing lipid transfer (1) between various model membrane assemblies (vesicles, micelles, bicelles, nanodiscs), (2) involving other lipid types by other lipid transfer proteins, (3) with protein preparations that are either crudely or highly purified, and (4) that is spontaneous and occurs in the absence of protein.
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References
Holthuis JCM, Menon AK (2014) Lipid landscapes and pipelines in membrane homeostasis. Nature 510:48–57
Drin G (2014) Topological regulation of lipid balance in cells. Annu Rev Biochem 83:51–77
Apodaca G, Brown WJ (2014) Membrane traffic research: challenges for the next decade. Front Cell Dev Biol 2:e52
Tatsuta T, Scharwey M, Langer T (2014) Mitochondrial lipid trafficking. Trends Cell Biol 24:44–52
Hurlock AK, Roston RL, Wang K et al (2014) Lipid trafficking in plant cells. Traffic 15:915–932
Malinina L, Simanshu DK, Zhai X et al (2015) Sphingolipid transfer proteins defined by the GLTP-fold. Q Rev Biophys 48:281–322
Yamaji T, Hanada K (2015) Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins. Traffic 16:101–122
Moser von Filseck J, Čopič A, Delfosse V et al (2015) Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate. Science 349:432–436
Hotamisligil GS, Bernlohr DA (2015) Metabolic functions of FABPs—mechanisms and therapeutic implications. Nat Rev Endocrinol 11:592–605
Grabon A, Khan D, Bankaitis VA (2015) Phosphatidylinositol transfer proteins and instructive regulation of lipid kinase biology. Biochim Biophys Acta 1851:724–735
Wakana Y, Kotake R, Oyama N et al (2015) CARTS biogenesis requires VAP–lipid transfer protein complexes functioning at the endoplasmic reticulum–Golgi interface. Mol Biol Cell 26:4686–4699
Mattjus P (2016) Specificity of the mammalian glycolipid transfer proteins. Chem Phys Lipids 194:72–78
Gallo A, Vannier C, Galli T (2016) Endoplasmic reticulum–plasma membrane associations: structures and functions. Annu Rev Cell Dev Biol 32:279–301
Tong J, Manik MK, Yang H et al (2016) Structural insights into nonvesicular lipid transport by the oxysterol binding protein homologue family. Biochim Biophys Acta 1861:928–939
Kentala H, Weber-Boyvat M, Olkkonen VM (2016) OSBP-related protein family: mediators of lipid transport and signaling at membrane contact sites. Int Rev Cell Mol Biol 321:299–340
Huang J, Ghosh R, Bankaitis VA (2016) Sec14-like phosphatidylinositol transfer proteins and the biological landscape of phosphoinositide signaling in plants. Biochim Biophys Acta 1861:1352–1364
Salminen TA, Blomqvist K, Edqvist J (2016) Lipid transfer proteins: classification, nomenclature, structure, and function. Planta 244:971–997
Wong LH, Levine TP (2016) Lipid transfer proteins do their thing anchored at membrane contact sites … but what is their thing? Biochem Soc Trans 44:517–527
de Campos MKF, Schaaf G (2017) The regulation of cell polarity by lipid transfer proteins of the SEC14 family. Curr Opin Plant Biol 40:158–168
Wong LH, Čopič A, Levine TP (2017) Advances on the transfer of lipids by lipid transfer proteins. Trends Biochem Sci 42:516–530
Malinina L, Patel DJ, Brown RE (2017) How α-helical motifs form functionally diverse lipid-binding compartments. Annu Rev Biochem 86:609–636
Huang J, Mousley CJ, Dacquay L et al (2018) A lipid transfer protein signaling axis exerts dual control of cell-cycle and membrane trafficking systems. Dev Cell 44:378–391
Mishra SK, Gao Y-G, Deng Y et al (2018) CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation. Autophagy 14(5):862–879
Malinina L, Malakhova ML, Teplov A et al (2004) Structural basis for glycosphingolipid transfer specificity. Nature 430:1048–1053
Kenoth R, Simanshu DK, Kamlekar RK et al (2010) Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP-fold. J Biol Chem 285:13066–13078
Simanshu DK, Kamlekar RK, Wijesinghe DS et al (2013) Nonvesicular trafficking of a ceramide-1-phosphate that regulates eicosanoids. Nature 500:463–468
Simanshu DK, Zhai X, Munch D et al (2014) Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels. Cell Rep 6:388–399
Ochoa-Lizarralde B, Gao Y-G, Popov AN et al (2018) How FAPP2 selects simple glycosphingolipids using the GLTP-fold: Structural insights into specificity. J Biol Chem (in press)
D'Angelo G, Polishchuk E, Di Tullio G et al (2007) Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide. Nature 449:62–67
Kamlekar RK, Simanshu DK, Gao YG et al (2013) The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids. Biochim Biophys Acta 1831:417–427
Bergelson LD, Molotkovsky JG, Manevich YM (1985) Lipid specific fluorescent probes in studies of biological membranes. Chem Phys Lipids 37:165–195
Molotkovsky JG, Mikhalyov II, Imbs AB et al (1991) Synthesis and characterization of new fluorescent glycolipid probes: molecular organization of glycosphingolipids in mixed composition lipid bilayer. Chem Phys Lipids 58:199–212
Boldyrev IA, Zhai X, Momsen MM et al (2007) New BODIPY lipid probes for fluorescence studies of membranes. J Lipid Res 48:1518–1532
Boldyrev IA, Brown RE, Molotkovsky JG (2013) An expedient synthesis of fluorescent labeled ceramide-1-phosphate analogues. Russ J Bioorgan Chem 39:539–542
Radin NS, Evangelatos GP (1981) The use of galactose oxidase in lipid labeling. J Lipid Res 22:536–541
Brown RE, Sugar IP, Thompson TE (1985) Spontaneous transfer of gangliotetraosylceramide between phospholipid vesicles. Biochemistry 24:4082–4091
Brown RE, Thompson TE (1987) Spontaneous transfer of ganglioside GM1 between phospholipid vesicles. Biochemistry 26:5454–5460
Sonnino S, Nicolini M, Chigorno V (1996) Preparation of radiolabeled gangliosides. Glycobiology 6:479–487
Sonnino S, Chigorno V, Tettamanti G (2000) Preparation of radioactive gangliosides, 3H or 14C isotopically labeled at oligosaccharide or ceramide moieties. Methods Enzymol 311:639–656
Schwarzmann G, Sandhoff K (1987) Lysogangliosides: synthesis and use in preparing labeled gangliosides. Methods Enzymol 138:319–341
Ito M, Mitsutake S, Tani M et al (2000) Enzymatic synthesis of [14C]ceramide, [14C]glycosphingolipids, and ω-aminoceramide. Methods Enzymol 311:682–689
Li X-M, Malakhova ML, Lin X et al (2004) Human glycolipid transfer protein: Probing conformation using fluorescence spectroscopy. Biochemistry 43:10285–10294
Malakhova ML, Malinina L, Pike HM et al (2005) Point mutational analysis of the liganding site in human glycolipid transfer protein: functionality of the complex. J Biol Chem 280:26312–26320
Szoka F, Demetrios Papahadjopoulos D (1980) Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu Rev Biophys Bioeng 9:467–508
Patil YP, Jadhav S (2013) Novel methods for liposome preparation. Chem Phys Lipids 177:8–18
Akbarzadeh A, Rezaei-Sadabady R, Davaran S et al (2014) Liposome: classification, preparation, and applications. Nanoscale Res Lett 8:e102
Nichols JW, Pagano RE (1981) Kinetics of soluble lipid monomer diffusion between vesicles. Biochemistry 20:2783–2789
Nichols JW (1988) Kinetics of fluorescent-labeled phosphatidylcholine transfer between nonspecific lipid transfer protein and phospholipid vesicles. Biochemistry 27:1889–1896
Roseman M, Thompson TE (1980) Mechanism of the spontaneous transfer of phospholipids between bilayers. Biochemistry 19:439–444
Correa-Freire MC, Barenholz Y, Thompson TE (1982) Glucocerebroside transfer between phosphatidylcholine bilayers. Biochemistry 21:1244–1248
Abe A, Sakagami T, Yamada K et al (1985) A fluorimetric determination of the activity of glycolipid transfer protein and some properties of the protein purified from pig brain. Biochim Biophys Acta 778:239–244
Wong M, Brown RE, Barenholz Y et al (1984) Glycolipid transfer protein from bovine brain. Biochemistry 23:6498–6505
Bai J, Pagano RE (1997) Measurement of spontaneous transfer and transbilayer movement of BODIPY-labeled lipids in lipid vesicles. Biochemistry 36:8840–8848
Elvington SM, Nichols JW (2007) Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs. Biochim Biophys Acta 1768:502–508
Mattjus P, Molotkovsky JG, Smaby JM et al (1999) A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes. Anal Biochem 268:297–304
Mattjus P, Pike HM, Molotkovsky JG et al (2000) Charged membrane surfaces impede the protein mediated transfer of glycosphingolipids between phospholipid bilayers. Biochemistry 39:1067–1075
Mattjus P, Kline A, Pike HM et al (2002) Probing for preferential interactions among sphingolipids in bilayer vesicles using the glycolipid transfer protein. Biochemistry 41:266–273
West G, Viitanen L, Alm C et al (2008) Identification of a glycosphingolipid transfer protein GLTP1 in Arabidopsis thaliana. FEBS J 275:3421–3437
Nylund M, Mattjus P (2005) Protein mediated glycolipid transfer is inhibited FROM sphingomyelin membranes but enhanced TO sphingomyelin containing raft like membranes. Biochim Biophys Acta 1669:87–94
Nylund M, Kjellberg MA, Molotkovsky JG et al (2006) Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles. Biochim Biophys Acta 1758:807–812
Nichols JW, Pagano RE (1983) Resonance energy transfer assay of protein-mediated lipid transfer between vesicles. J Biol Chem 258:5368–5371
Schwarzmann G, Wendeler M, Sandhoff K (2005) Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system. Glycobiology 15:1302–1311
Epand RF, Schlattner U, Wallimann T et al (2007) Novel lipid transfer property of two mitochondrial proteins that bridge the inner and outer membranes. Biophys J 92:126–137
Brown RE (1990) Spontaneous transfer of lipids between membranes. Subcell Biochem 16:333–363
Jones JD, Almeida PFF, Thompson TE (1990) Spontaneous interbilayer transfer of hexosylceramides between phospholipid bilayers. Biochemistry 29:3892–3897
Brown RE (1992) Spontaneous lipid transfer between organized lipid assemblies. Biochim Biophys Acta 1113:375–389
Brown RE, Mattjus P (2007) Glycolipid transfer proteins. Biochim Biophys Acta 1771:746–760
Rao CS, Lin X, Pike HM et al (2004) Glycolipid transfer protein mediated transfer of glycosphingolipids between membranes: a model for action based on kinetics and thermodynamic analyses. Biochemistry 43:13805–13815
Samygina VR, Popov AN, Cabo-Bilbao A et al (2011) Structure 19:1644–1654
Brown RE, Stephenson FA, Markello T et al (1985) Properties of a specific glycolipid transfer protein from bovine brain. Chem Phys Lipids 38:79–93
Brown RE, Jarvis KL, Hyland KJ (1990) Purification and characterization of glycolipid transfer protein from bovine brain. Biochim Biophys Acta 1044:77–83
Zhai X, Gao Y-G, Mishra SK et al (2017) Phosphatidylserine stimulates ceramide 1-phosphate (C1P) intermembrane transfer by C1P transfer proteins. J Biol Chem 292:2531–2541
Brown RE, Hyland KJ (1992) Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size. Biochemistry 31:10602–10609
Lev S (2010) Non-vesicular lipid transport by lipid-transfer proteins and beyond. Nat Rev Mol Cell Biol 11:739–750
Somerharju P (2015) Is spontaneous translocation of polar lipids between cellular organelles negligible? Lipid Insights 8(S1):87–93
Richens JL, Tyler AII, Barriga HMG et al (2017) Spontaneous charged lipid transfer between lipid vesicles. Sci Report 7:e12606
Shenkarev ZO, Melnikova DN, Finkina EI et al (2017) Ligand binding properties of the lentil lipid transfer protein: Molecular insight into the possible mechanism of lipid uptake. Biochemistry 56:1785–1796
Sumi M, Makino A, Inaba T et al (2017) Photoswitchable phospholipid FRET acceptor: detergent free intermembrane transfer assay of fluorescent lipid analogs. Sci Report 7:e2900
Hölttä-Vuori M, Uronen R-L, Repakova J et al (2008) BODIPY-Cholesterol: A new tool to visualize sterol trafficking in living cells and organisms. Traffic 9:1839–1849
Locatelli-Hoops S, Remmel N, Klingenstein R et al (2006) Saposin a mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: Patient variant saposin A lacks lipid extraction capacity. J Biol Chem 281:32451–32460
Kernstock RM, Girotti AW (2007) Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology. Anal Biochem 365:111–121
Sugiki T, Takahashi H, Nagasu M et al (2010) Real-time assay method of lipid extraction activity. Anal Biochem 399:162–167
Ohvo-Rekilä H, Mattjus P (2011) Monitoring glycolipid transfer protein activity and membrane interaction with the surface plasmon resonance technique. Biochim Biophys Acta 1808:47–54
Davison JM, Bankaitis VA, Ghosh R (2012) Devising powerful genetics, biochemical and structural tools in the functional analysis of phosphatidylinositol transfer proteins (PITPs) across diverse species. Methods Cell Biol 108:249–302
Hanada K, Sugiki T (2017) In vitro assay to extract specific lipid types from phospholipid membranes using lipid-transfer proteins: a lesson from the ceramide transport protein CERT. In: Wood P (ed) Lipidomics, Neuromethods, vol 125. Humana Press, New York, NY
Zhai X, Malakhova ML, Pike HP et al (2009) Glycolipid acquisition by human glycolipid transfer Protein dramatically alters intrinsic tryptophan fluorescence: Insights into glycolipid binding affinity. J Biol Chem 284:13620–13628
Kamlekar RK, Gao Y-G, Kenoth R et al (2010) Human GLTP: three distinct functions for the three tryptophans in a novel peripheral amphitropic fold. Biophys J 99:2626–2635
Kenoth R, Zou X, Simanshu DK et al (2018) Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein. BBA-Biomembranes 1860:1069–1076
Zhai X, Momsen WE, Malakhov DA et al (2013) GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine. J Lipid Res 54:1103–1113
Kremer JM, Esker MW, Pathmamanoharan C, Wiersema PH (1977) Vesicles of variable diameter prepared by a modified injection method. Biochemistry 16:3932–3935
Nordlund JR, Schmidt CF, Dicken SN et al (1981) Transbilayer distribution of phosphatidylethanolamine in large and small unilamellar vesicles. Biochemistry 20:3237–3241
Schwarzmann G, Breiden B, Sandhoff K (2015) Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. J Lipid Res 56:1861–1879
Coldren B, van Zanten R, Mackel MJ et al (2003) From vesicle size distributions to bilayer elasticity via cryo-transmission and freeze-fracture electron microscopy. Langmuir 19:5632–5639
Meister A, Blume A (2017) (Cryo)transmission electron microscopy of phospholipid model membranes interacting with amphiphilic and polyphilic molecules. Polymers 9:521
Edqvist J, Rönnberg E, Rosenquist S et al (2004) Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2. J Biol Chem 279:53544–53553
Wetterau JR, Zilversmit DB (1984) Quantitation of lipid transfer activity. Methods Biochem Anal 30:199–226
Acknowledgments
We are grateful to many individuals who contributed to the development of the experimental approaches used routinely in the REB lab for many years and detailed here. They include J.G. Molotkovsky, H.M. Pike, X Zhai, I.A. Boldyrev, Y-G Gao, and P. Mattjus. We also are grateful for support from Dept. of Science and Technology, Science and Engineering Research Board (SERB), Govt. of India to RKK (YSS/2014/000021) and to RK (YSS/2015/000783) as well as support by NIH/NIGMS-GM45928, NIH/NCI-CA121493, and NIH/NHLBI-HL125353 (to REB) and the Hormel Foundation. We thank VIT for research facilities and infrastructure.
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Kenoth, R., Brown, R.E., Kamlekar, R.K. (2019). In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_17
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