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Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

量化检测纯化Drp1与合成脂质体之间的相互作用

YA Yoshihiro Adachi
KI Kie Itoh
MI Miho Iijima
HS Hiromi Sesaki

发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2266 浏览次数: 11363

评审: Nicoletta CordaniJianwei SunDaniel Kraus

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Abstract

A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.

Keywords: Mitochondria (线粒体) search Organelle division (细胞器分裂) search Dynamin superfamily (动力蛋白超家族) search Phospholipids (磷脂) search

Background

Interactions of proteins and membrane lipids are critical for the remodeling of biological membranes in cells such as organelle division. In mitochondrial division, Drp1 constricts the mitochondrial membranes and drives this membrane remodeling process. We have recently shown that a signaling phospholipid, phosphatidic acid, interacts with Drp1 and creates the priming step by restraining assembled division machinery on mitochondria (Adachi et al., 2016). Drp1 recognizes both the head group and acyl chains of phosphatidic acid. To analyze Drp1-phosphatidic acid binding, we set up several protein-lipid interaction assays, including a lipid dot blot assay, a competition assay and a liposome flotation assay. These assays allowed us to determine the function of the head group and acyl chain composition in protein-lipid interactions. In addition, we analyzed lipids under bilayer conditions in the liposome assay and lipids under non-bilayer conditions in dot blot and competition assays, which made it possible to examine the contribution of the membrane curvature and lipid packing to protein-lipid interactions. Below, we describe a liposome flotation assay that can be applied to many peripheral membrane proteins.

Materials and Reagents

  1. 0.45 µm Millex-HA syringe filter unit (EMD Millipore, catalog number: SLHA033SS )
  2. 15-ml column (Poly-prep chromatography column) (Bio-Rad Laboratories, catalog number: 7311550 )
  3. Amicon Ultra-15 centrifugal filter unit with Ultracel-10 membrane (10k filter for domains of Drp1) (EMD Millipore, catalog number: UFC901024 )
  4. Amicon Ultra-15 centrifugal filter unit with Ultracel-50 membrane (50k filter for full length Drp1) (EMD Millipore, catalog number: UFC905024 )
  5. Polycarbonate membranes 0.4 μm (Avanti Polar Lipids, catalog number: 610007 )
  6. 96 well assay plate (Corning, catalog number: 3915 )
  7. Extruder set with holder/heating block (Avanti Polar Lipids, catalog number: 610000 )
  8. Filter support (Avanti Polar Lipids, catalog number: 610014 )
  9. 2 ml amber glass sample vials (WHEATON, catalog number: 224981 )
  10. Disposable culture tubes (Fisher Scientific, catalog number: 14-961-27 )
  11. 50 Ulrta-Clear tubes (Beckman Coulter, catalog number: 344057 )
  12. RosettaTM 2(DE3)pLysS competent cells (EMD Millipore, catalog number: 71403 )
  13. pET15b vectors (EMD Millipore, Novagen)
  14. 50% Ni-NTA beads (EMD Millipore, catalog number: 70666 )
  15. 4-20% Tris-HCl precast gel (Bio-Rad Laboratories, catalog number: 3450034 )
  16. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  17. Ethanol (PHARMCO-AAPER, catalog number: 111000200 )
  18. DMSO (Fisher Scientific, catalog number: S369-500 )
  19. Liquid nitrogen
  20. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457 )
  21. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-DPPE) (Avanti Polar Lipids, catalog number: 810158 )
  22. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) (Avanti Polar Lipids, catalog number: 850705 )
  23. 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) (Avanti Polar Lipids, catalog number: 830855 )
  24. 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (Avanti Polar Lipids, catalog number: 840875 )
  25. 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (Palmitoyl lysoPA) (Avanti Polar Lipids, catalog number: 857123 )
  26. 1,2-dihexanoyl-sn-glycero-3-phosphate (Dihexanoyl PA) (Avanti Polar Lipids, catalog number: 830841 )
  27. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Avanti Polar Lipids, catalog number: 850355 )
  28. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, catalog number: 850375 )
  29. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti Polar Lipids, catalog number: 850725 )
  30. 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DPPG) (Avanti Polar Lipids, catalog number: 840455 )
  31. 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG) (Avanti Polar Lipids, catalog number: 840475 )
  32. 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) (Avanti Polar Lipids, catalog number: 840037 )
  33. 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (Avanti Polar Lipids, catalog number: 840035 )
  34. 1’,3’-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL) (Avanti Polar Lipids, catalog number: 710335 )
  35. 1’,3’-bis[1,2-dipalmitoyl-sn-glycero-3-phospho]-sn-glycerol TPCL (Echelon Biosciences, catalog number: L-C160 )
  36. Gas nitrogen
  37. SilverQuestTM Silver Staining Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC6070 )
  38. BactoTM tryptone (BD, BactoTM, catalog number: 211705 )
  39. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
  40. BactoTM yeast extract (BD, BactoTM, catalog number: 212750 )
  41. Agar (Alfa Aesar, catalog number: J10906 )
  42. Ultrapure water (e.g., MilliQ)
  43. Ampicillin, sodium salt (Alfa Aesar, catalog number: J11259 )
  44. Chloramphenicol (Mediatech, catalog number: 61-239-RI )
  45. beta-D-Galactopyranoside, Isopropyl-beta-D-thiogalactopyranoside (IPTG) (AMRESCO, catalog number: 0487 )
  46. HEPES (Fisher Scientific, catalog number: BP310-1 )
  47. Potassium hydroxide (KOH) (Avantor® Performance Materials, J.T. Baker, catalog number: 3116-01 )
  48. Magnesium chloride hydrate (MgCl2·6H2O) (Avantor® Performance Materials, J.T. Baker, catalog number: 2444 )
  49. Imidazole (Sigma-Aldrich, catalog number: I0125 or I202 )
    Note: The product “ I0125 ” has been discontinue
  50. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  51. Sodium phosphate monobasic (NaH2PO4·H2O) (Fisher Scientific, catalog number: S369-500 )
  52. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: S373-3 )
  53. Sodium hydroxide (NaOH) (Avantor® Performance Materials, J.T. Baker, catalog number: 3722 )
  54. Coomassie Brilliant Blue R-250 (AMRESCO, catalog number: M128-50G )
  55. Acetic acid (Fisher Scientific, catalog number: A38-212 )
  56. Methanol (Fisher Scientific, catalog number: A412 )
  57. MES (2-morpholinoethanesulfonic acid, mono hydrate) (Sigma-Aldrich, catalog number: M8250 )
  58. Sucrose (Fisher Scientific, catalog number: S5-3 )
  59. LB plate containing ampicillin and chloramphenicol (see Recipes)
  60. LB with ampicillin and chloramphenicol (see Recipes)
  61. 0.5 M IPTG
  62. 0.5 M HEPES (pH 7.4) (see Recipes)
  63. Lysis buffer (see Recipes)
  64. Wash buffer (see Recipes)
  65. Elution buffer (see Recipes)
  66. 10x PBS (see Recipes)
  67. Coomassie Brilliant Blue solution (see Recipes)
  68. 100 mM MES (pH 7.0) (see Recipes)
  69. 20 mM MES (pH 7.0) (see Recipes)
  70. 20 mM MES (pH 7.0)/100 mM NaCl (see Recipes)
  71. 2.7 M sucrose/20 mM MES/100 mM NaCl (see Recipes)
  72. 1.25 M sucrose/20 mM MES/100 mM NaCl (see Recipes)
  73. 0.25 M sucrose/20 mM MES/100 mM NaCl (see Recipes)

Equipment

  1. 1 ml syringe (Hamilton, catalog number: 81365 )
  2. 500 µl syringe (Hamilton, catalog number: 81265 )
  3. 100 µl syringe (Hamilton, catalog number: 81065 )
  4. 50 µl syringe (Hamilton, catalog number: 80500 )
  5. 25 µl syringe (Hamilton, catalog number: 80400 )
  6. Incubator shaker (37 °C) (Eppendorf, New BrunswickTM, model: Series 25 )
  7. Incubator shaker (16 °C) (Lab Companion, model: IS-971R )
  8. 1,000 ml bottle (Beckman Coulter, catalog number: A98814 )
  9. JLA8.1000 rotor (Beckman Coulter, catalog number: 969329 )
  10. GH3.8 rotor (Beckman Coulter, catalog number: 360581 )
  11. Fisher Scientific Sonic dismembrator (Fisher Scientific, model: 100 )
  12. CS-6R centrifuge (Beckman Coulter, model: CS-6R )
  13. Avanti J-E (Beckman Coulter, model: Avanti J-E , catalog number: 369001)
  14. SpeedVac SVC100 (Savant System, model: SpeedVac SVC100 )
  15. Heat block (37 °C, 42 °C, 95 °C) (Fisher Scientific, model: Fisher ScientificTM IsotempTM Digital and Analog Dry Bath Incubators , catalog number: 11-718)
  16. Motorized upright fluorescence microscope (Olympus, model: BX61 )
  17. POLARstar Omega (BMG LABTECH, model: POLARstar Omega )
  18. Labquake Shaker Rotisserie (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4152110 )
  19. SW55Ti rotor (Beckman Coulter, catalog number: 342196 )
  20. Avanti J-26XP (Beckman Coulter, model: Avanti® J-26 XP , catalog number: 393124)
  21. Optima L-100XP ultracentrifuge (Beckman Coulter, model: OptimaTM L-100XP , catalog number: 392050)
  22. Scanmaker 8700 (Microtek, model: Scanmaker 8700 )
  23. Power supply (Bio-Rad Laboratories, model: PowerPacTM HC )
  24. Multi-Purpose rotator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2309 )
  25. Vortex mixer (Fisher Scientific, model: Fisher ScientificTM Analog Vortex Mixer , catalog number: 02215365)

Software

  1. ImageJ (Version 1.48, http://imagej.net/)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Adachi, Y., Itoh, K., Iijima, M. and Sesaki, H. (2017). Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes. Bio-protocol 7(9): e2266. DOI: 10.21769/BioProtoc.2266.

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分类

生物化学 > 脂质 > 脂质-蛋白互作

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