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Liposome Flotation Assays for Phosphoinositide-protein Interaction
磷酸肌醇-蛋白质相互作用的脂质体浮选分析法   

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Abstract

Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different subcellular compartments (Di Paolo and De Camilli, 2006; Lemmon, 2003). Therefore, analysis of these effectors’ binding properties and specificity towards different phosphoinositides is crucial for the understanding of their cellular functions. This protocol describes a method to characterize the binding of proteins to different phosphoinositide-containing vesicles.

Keywords: Liposome flotation assay(脂质体浮选分析法), Phosphoinositides(磷酸肌醇), Protein-lipid interaction(蛋白质-脂质相互作用), Unilamellar vesicles(单层囊泡)

Background

Several methods exist to analyze protein-phosphoinositide binding and the specificity towards the different members of the phosphoinositide family: lipid overlay, lipid flotation assay and surface plasma resonance (SPR). A Lipid flotation assay consists in the incubation of unilamellar vesicles with the protein of interest, and the subsequent flotation of the vesicles on a sucrose cushion, which will separate vesicle-bound proteins from unbound proteins and vesicles alone. Compared to the other methods a lipid flotation assay is i) technically easier and cheaper than SPR and ii) more specific and closer to physiological conditions, as it mimics the curvature of membranes, in contrast to protein-overlay assays, where lipids are dried on a flat nitrocellulose membrane. This protocol describes the binding of recombinant GST-tagged proteins to unilamellar vesicles of defined size and lipid composition, in particular regarding the specificity towards the different monophosphorylated phosphoinositides (PIP).

Materials and Reagents

  1. Glass tubes, 12 x 75 mm (DUTSCHER SCIENTIFIC, catalog number: 110011 )
  2. Tube, thickwall, polycarbonate, 1 ml, 11 x 34 mm (Beckman Coulter, catalog number: 343778 )
  3. Stericup-GP sterile vacuum filter unit, polyethersulfone, 0.22 μm (EMD Millipore, catalog number: SCGPU10RE )
  4. Mini Extruder Kit (mini-extruder, 2 Hamilton syringes, 0.1 μm polycarbonate membranes, filter supports, holder/heating block) (Avanti Polar Lipids, catalog number: 610000 )
  5. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) (Avanti Polar Lipids, catalog number: 850457 )
  6. 1-palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (PE) (Echelon, catalog number: L-2368 )
  7. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (fluoPE) (Avanti Polar Lipids, catalog number: 810332 )
  8. Phosphatidylinositol 3-phosphate diC16 (PI3P) (Echelon, catalog number: P3016 )
  9. L-α-phosphatidylinositol-4-phosphate (PI4P) from porcine brain (Avanti Polar Lipids, catalog number: 840045 )
  10. Phosphatidylinositol 5-phosphate diC16 (PI5P) (Echelon, catalog number: P5016 )
  11. Liquid nitrogen
  12. Chloroform/methanol (50/50, v/v)
  13. PBS (w/o Ca/Mg) (Sigma-Aldrich, catalog number: D8537 )
  14. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  15. Anti-GST antibody (clone B-14) (Santa Cruz Biotechnology, catalog number: sc-138 )
  16. Lipid stocks solutions (see Recipes)
  17. 60% and 25% sucrose solutions in PBS (see Recipes)

Equipment

  1. Nitrogen evaporator (N-EVAP, Organomation, catalog number: 11250 )
  2. Chemical hood
  3. Standard heated water bath
  4. Optima TLX ultracentrifuge (Beckman Coulter, model: OptimaTM TLX)
  5. TLA100.2 rotor (Beckman Coulter, model: TLA100.2 rotor)
    Note: This rotor is not available anymore from Beckman Coulter. Rotor TLA120.2 (Beckman Coulter, model: TLA120.2) can be used instead.
  6. Portable UV lamp
  7. SDS-PAGE running apparatus
  8. Western blotting apparatus
  9. Refractometer

Software

  1. Image analysis software (e.g., ImageJ, NIH)

Procedure

  1. Prepare the large unilamellar vesicles (LUVs)
    1. To prepare the lipids stock solutions (see Recipes). When using fluorescently labeled lipids, shield from direct light throughout the procedure.
    2. Prepare a 2x concentrated lipid mix at 1 mM concentration in a glass tube: PC:PE:fluoPE (70:28:2 mol%) to assess basal binding properties and PC:PE:fluoPE:PIP (64:28:2:6 mol%) to assess specific binding properties of the protein to each PIP.
    3. Evaporate under nitrogen.
    4. Resuspend twice with chloroform/methanol (v/v) and evaporate under nitrogen to form a nice lipid cake on the glass tube wall. No clumps should remain at this stage.
    5. Evaporate under nitrogen for a final 30 min-1 h to eliminate any traces of solvent.
    6. Resuspend in 37 °C pre-warmed PBS and leave to rehydrate for 1 h at RT.
    7. Perform 5 freeze/thaw cycles in liquid nitrogen/warm water (40 °C). For optimal results, wait for complete freezing/thawing of the samples for each step. Performing these freeze-thaw cycles will help to break the lipid cake and will ensure a good efficiency of the extruder in the following step, therefore improving the results. We found that 5 cycles was providing the best reliability of the results without being too much time-consuming.
    8. Mount the mini-extruder according to manufacturer’s instructions using the appropriate membrane size (e.g., 0.1 μm polycarbonate membrane for LUVs formation).
    9. Extrude the lipid mix through the filter, typically 19 times. Collect and transfer into a fresh glass tube.

  2. LUVs-protein mix
    1. In a thick-wall polycarbonate tube, mix 50 µl of the 2x LUVs with 50 µl of the 2x GST-recombinant protein to have a final volume of 100 µl and a final concentration 0.5 mM LUVs and 1 mM recombinant protein. See Notes section for adaptability. Include as negative control the purified GST protein alone with the LUVs.
    2. Incubate for 1 h at RT.
    3. Add 100 μl of 60% sucrose in PBS to yield a 30% final sucrose concentration. Mix gently to have a homogenous solution but without disrupting the interaction. See Figure 1 for a schematic representation.
    4. Overlay 250 μl of 25% sucrose and 50 μl of PBS buffer on top without disturbing the layers.
    5. Centrifuge at 174,206 x g for 1 h at 20 °C (corresponding to 70,000 rpm on a TLA100.2 rotor). In order not to disturb the layers and to get reliable results, it is important to use slower acceleration/deceleration parameters than usual, e.g., acceleration 2/deceleration 2 on an OptimaTM TLX ultracentrifuge.


      Figure 1. Schematic representation of the liposome binding assay. LUVs and protein mix equilibrated in 30% sucrose are layered with 25% and 0% sucrose, centrifuged to allow LUVs flotation (70 000 rpm [174,206 x g] on a TLA100.2 rotor). Fractions are collected with Hamilton’s syringe from the bottom of the tube.

  3. Collect the fractions
    1. As the liposomes will fluoresce owing to the fluo-PE, collect the fractions under a portable UV lamp using a Hamilton syringe, starting from the bottom of the tube (see Figure 1 for schematic representation).
      1. 200 μl bottom fraction
      2. 200 μl middle fraction
      3. 100 μl top fraction containing the LUVs and associated proteins.

Data analysis

Mix 20 µl of the top fraction with sample buffer and resolve by SDS-PAGE. Analyze the amount of recombinant proteins bound to the LUVs by either Coomassie stain or Western blot against the GST moiety or using a specific antibody. Band intensity can be quantified by densitometry using an image analysis software (e.g., ImageJ, NIH) in order to compare binding specificity towards the different PIP. Typically, the GST tag alone does not bind to LUVs, but this has to be checked as a negative control. As shown in Figure 2, we previously used this technique in order to analyze the PIP binding specificity of different structural domains of the endosomal protein TOM1 (Boal et al., 2015). We demonstrated that the VHS domain shows a greater avidity for PI5P, in contrast to the GAT domain (Figure 2B).


Figure 2. TOM1 VHS but not GAT domain shows specificity towards PI5P binding in a lipid flotation assay. A. TOM1 structural domains; B. N-terminally GST- tagged VHS or GAT domains were incubated with liposomes containing 6% mol of the indicated PIP. After flotation on a sucrose cushion, bound proteins were analyzed by Western blot using an anti-GST antibody (left panel). Right panel shows quantification across 4 independent experiments. Results are presented as mean ± SEM; t-test *P < 0.05; ns, non-significant.

Notes

  1. Once prepared, LUVs stock solution can be kept in a nitrogen-filled sealed glass tube at 4 °C for a couple of days but one might expect some oxidation and should take care when analyzing the results.
  2. According to the affinity of the protein towards the LUVs, the final concentration for the recombinant protein can be modified. Similarly, composition of the binding buffer can be adapted, e.g., buffer specificity, calcium-dependence, blocking agent (fatty-acid free BSA).
  3. Centrifugation step should be performed with gentle acceleration/deceleration in order not to disturb the sucrose layers.
  4. The use of fluoPE allows a quick and easy visualization of the flotation. When collecting the fractions, one must take care not to disturb the top layer, as that will induce cross-contamination and reduce reproducibility of the results.
  5. The purity of the recombinant protein is crucial. Indeed, bacterial contaminants could greatly alter the results. Purity must at least be checked by SDS-PAGE followed by Coomassie stain. Protein aggregates must be eliminated by centrifugation at full speed. In case of affinity-purification using the GST-tag, the elution buffer must be exchanged for the binding buffer (e.g., PBS) by dialysis.

Recipes

  1. Lipid stocks solutions
    Prepared in chloroform/methanol at the concentration of 1 mg/ml in glass tubes
    Stored at -20 °C
    Note: After each use, apply a stream of nitrogen to fill in the glass tube and avoid lipid oxidation but limit evaporation of chloroform/methanol as this will modify the concentration of the stocks. Tighten the cap with Teflon rubber.
  2. 60% and 25% sucrose solutions in PBS
    Dissolve the sucrose in PBS (60% eq. 771.9 g/L and 25% eq. 275.9 g/L)
    Once fully dissolved, check density with a refractometer and adjust with PBS
    Note: As sucrose density fluctuates with temperature, take care to fully equilibrate your buffer. Filtrate with Millipore stericup sterile vacuum filter unit. In order to prevent bacterial contamination, the sucrose stock solutions have to be kept sterile and at 4 °C.

Acknowledgments

This protocol was modified from the work of Bigay and Antonny (2005). This study was supported by grants from Institut National de la Santé et de la Recherche Médicale; Agence Nationale de la Recherche; Fondation pour la Recherche Médicale; The French Muscular Dystrophy Association (AFM); and by the Centre National de la Recherche Scientifique; the Région Midi-Pyrénées and European funds (FEDER, Fonds Européens de Développement Régional). The authors declare no conflict of interest or competing interests.

References

  1. Bigay, J. and Antonny, B. (2005). Real-time assays for the assembly-disassembly cycle of COP coats on liposomes of defined size. Methods Enzymol 404: 95-107.
  2. Boal, F., Mansour, R., Gayral, M., Saland, E., Chicanne, G., Xuereb, J. M. and Tronchère, H. (2015). TOM1 is a PI5P effector involved in the regulation of endosomal maturation. J Cell Sci 128(4): 815-827.
  3. Di Paolo, G. and De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature 443(7112): 651-657.
  4. Lemmon, M. A. (2003). Phosphoinositide recognition domains. Traffic 4(4): 201-213.

简介

磷酸肌醇是涉及控制主要细胞功能和信号通路的稀有膜脂质。他们能够将特异性效应物蛋白募集到质膜和细胞器的胞质表面,以协调各种信号传递和转运过程,并保持不同亚细胞区室的特定身份(Di Paolo和De Camilli,2006; Lemmon, 2003)。因此,这些效应物对不同磷酸肌醇的结合特性和特异性的分析对于了解其细胞功能至关重要。该方案描述了表征蛋白质与不同含磷酸肌醇的囊泡结合的方法。

背景 分析蛋白质 - 磷酸肌醇结合和对磷酸肌醇家族不同成员的特异性的几种方法:脂质覆盖,脂质浮选测定和表面等离子体共振(SPR)。脂质浮选测定法包括将单层囊泡与目的蛋白质孵育,随后在蔗糖垫上浮选囊泡,其将单独的囊泡结合的蛋白质与未结合的蛋白质和囊泡分离。与其他方法相比,脂质浮选测定是i)在技术上比SPR更容易和更便宜,ii)与蛋白质重叠测定相比,更具体和更接近于生理条件,因为它模拟膜的曲率,其中脂质被干燥平的硝酸纤维素膜。该方案描述了重组GST标记的蛋白质与确定大小和脂质组成的单层囊泡的结合,特别是关于对不同单磷酸化磷酸肌醇(PIP)的特异性。

关键字:脂质体浮选分析法, 磷酸肌醇, 蛋白质-脂质相互作用, 单层囊泡

材料和试剂

  1. 玻璃管,12 x 75毫米(DUTSCHER SCIENTIFIC,目录号:110011)
  2. 管,厚壁,聚碳酸酯,1ml,11×34mm(Beckman Coulter,目录号:343778)
  3. Stericup-GP无菌真空过滤器,聚醚砜0.22μm(EMD Millipore,目录号:SCGPU10RE)
  4. 迷你挤出机套件(迷你挤出机,2个Hamilton注射器,0.1μm聚碳酸酯膜,过滤器支架,保持器/加热块)(Avanti Polar Lipids,目录号:610000)
  5. 1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱(PC)(Avanti Polar Lipids,目录号:850457)
  6. 1-棕榈酰-3-油酰基-sn-甘油基-2-磷酸乙醇胺(PE)(Echelon,目录号:L-2368)
  7. 1,2-二油酰-sn-甘油-3-磷酸乙醇胺-N-(羧基荧光素)(fluoPE)(Avanti Polar Lipids,目录号:810332)
  8. 磷脂酰肌醇3-磷酸diC16(PI3P)(Echelon,目录号:P3016)
  9. 来自猪脑的L-α-磷脂酰肌醇-4-磷酸(PI4P)(Avanti Polar Lipids,目录号:840045)
  10. 磷脂酰肌醇5-磷酸diC16(PI5P)(Echelon,目录号:P5016)
  11. 液氮
  12. 氯仿/甲醇(50/50,v/v)
  13. PBS(w/o Ca/Mg)(Sigma-Aldrich,目录号:D8537)
  14. 蔗糖(Sigma-Aldrich,目录号:S0389)
  15. 抗GST抗体(克隆B-14)(Santa Cruz Biotechnology,目录号:sc-138)
  16. 脂质储备溶液(见配方)
  17. PBS中的60%和25%蔗糖溶液(参见食谱)

设备

  1. 氮气蒸发器(N-EVAP,Organomation,目录号:11250)
  2. 化学罩
  3. 标准加热水浴
  4. Optima TLX超速离心机(Beckman Coulter,型号:Optima TM TLX)
  5. TLA100.2转子(Beckman Coulter,型号:TLA100.2转子)
    注意:该转子不再可以从Beckman Coulter获得。转子TLA120.2(Beckman Coulter,型号:TLA120.2)可以代替。
  6. 便携式紫外线灯
  7. SDS-PAGE运行装置
  8. 蛋白质印迹装置
  9. 折射仪

软件

  1. 图像分析软件(例如,,ImageJ,NIH)

程序

  1. 准备大单层囊泡(LUVs)
    1. 准备脂质储备溶液(参见食谱)。当使用荧光标记的脂质时,在整个过程中屏蔽直射光
    2. 在玻璃管中制备1mM浓度的2x浓缩脂质混合物:PC:PE:fluoPE(70:28:2mol%)以评估基础结合性质和PC:PE:fluoPE:PIP(64:28:2:6 mol%)以评估蛋白质对每个PIP的特异性结合特性
    3. 在氮气下蒸发
    4. 用氯仿/甲醇(v/v)重悬二次,在氮气下蒸发,在玻璃管壁上形成不错的脂质饼。在这个阶段,不应该留下任何一块。
    5. 在氮气下蒸发最后30分钟-1小时以消除任何痕量的溶剂
    6. 重悬于37℃预热的PBS中,并在室温下放置1小时。
    7. 在液氮/温水(40°C)中进行5次冻融循环。为获得最佳效果,请等待每个步骤的样品完全冷冻/解冻。进行这些冻融循环将有助于破坏脂质滤饼,并确保挤出机在以下步骤中的良好效率,从而改善结果。我们发现5个周期提供了最佳的结果可靠性,而不需要花费太多时间
    8. 根据制造商的说明使用适当的膜尺寸(例如,用于LUV形成的0.1μm聚碳酸酯膜)安装小型挤出机。
    9. 通过过滤器将脂质混合物挤出,通常为19次。收集并转移到新鲜的玻璃管中
  2. LUVs蛋白质混合物
    1. 在厚壁聚碳酸酯管中,将50μl的2xLUV与50μl的2xGST重组蛋白质混合,使终体积为100μl,最终浓度为0.5mM LUV和1mM重组蛋白。有关适应性,请参见注释部分。将纯化的GST蛋白作为阴性对照包含在LUV中
    2. 在室温下孵育1小时。
    3. 在PBS中加入100μl60%蔗糖,得到30%的最终蔗糖浓度。轻轻混合以获得均匀的溶液,但不破坏相互作用。参见图1的示意图。
    4. 在顶部叠加250μl的25%蔗糖和50μl的PBS缓冲液,而不会扰乱层。
    5. 在204℃离心,在20℃下(相当于TLA100.2转子上的70,000rpm)1小时。为了不扰乱层数并获得可靠的结果,使用比平常更慢的加速/减速参数(例如),Optima TM上的加速度2 /减速度2 < sup> TLX超速离心机。


      图1.脂质体结合测定的示意图。在30%蔗糖中平衡的LUV和蛋白质混合物用25%和0%蔗糖分层,离心以允许LUV漂浮(70,000rpm [174,206 > xg ]在TLA100.2转子上)。从Hamilton的注射器从管的底部收集级分。

  3. 收集分数
    1. 由于脂质体会由于fluo-PE而发荧光,使用汉密尔顿注射器在便携式UV灯下收集部分,从管的底部开始(参见图1的示意图)。
      1. 200μl底部分数
      2. 200μl中间分数
      3. 100μl含有LUVs和相关蛋白的顶级成分。

数据分析

将20μl顶部馏分与样品缓冲液混合,并通过SDS-PAGE分析。通过考马斯染色法或蛋白印迹法分析与GST部分或使用特异性抗体结合LUVs的重组蛋白质的量。可以使用图像分析软件(例如,ImageJ,NIH)通过光密度测定来测量带强度,以便比较不同PIP的结合特异性。通常,单独的GST标签不与LUV结合,但是必须将其作为阴性对照进行检查。如图2所示,我们以前使用这种技术来分析内质蛋白TOM1的不同结构域的PIP结合特异性(Boal等,2015)。与GAT域相比,我们证明了VHS域对PI5P的亲和力更高(图2B)。


图2.TOM1VHS但不是GAT结构域显示在脂质浮选测定中对PI5P结合的特异性。
A.TOM1结构域; B.将N末端GST标记的VHS或GAT结构域与含有6%mol所示PIP的脂质体孵育。在蔗糖垫上漂浮后,使用抗GST抗体(左图)通过蛋白质印迹分析结合的蛋白质。右侧面板显示了4个独立实验的量化。结果以平均值±SEM表示; -test * P 0.05; ns,不重要。

笔记

  1. 一旦准备好,LUVs储备溶液可以在4℃的氮气密封的玻璃管中保存几天,但是可能会预期一些氧化,并且在分析结果时应该小心。
  2. 根据蛋白质对LUV的亲和力,可以修饰重组蛋白的最终浓度。类似地,结合缓冲液的组成可以适应,例如缓冲液特异性,钙依赖性,封闭剂(无脂肪酸的BSA)。
  3. 离心步骤应以温和的加速/减速进行,以免干扰蔗糖层
  4. 使用fluoPE可以快速简便地显示浮选。收集分数时,必须注意不要打扰顶层,否则会引起交叉污染并降低结果的重复性。
  5. 重组蛋白的纯度至关重要。事实上,细菌污染物可能会大大改变结果。至少必须通过SDS-PAGE检测纯度,然后进行考马斯染色。必须通过全速离心来消除蛋白质聚集体。在使用GST标签进行亲和纯化的情况下,必须通过透析将洗脱缓冲液更换为结合缓冲液(例如PBS)。

食谱

  1. 脂质储备解决方案
    在氯仿/甲醇中以1mg/ml的浓度在玻璃管中制备
    储存于-20°C 注意:每次使用后,应用氮气流填充玻璃管,避免脂质氧化,但限制氯仿/甲醇的蒸发,因为这将改变库存的浓度。用Teflon橡胶拧紧盖子。
  2. PBS中的60%和25%蔗糖溶液
    将蔗糖溶于PBS(60%eq。771.9g/L和25%eq。275.9g/L)中。 一旦完全溶解,用折射计检查密度并用PBS调整
    注意:随着蔗糖浓度随温度而变化,请注意使缓冲液完全平衡。用Millipore stericup无菌真空过滤器过滤。为了防止细菌污染,蔗糖储备溶液必须保持无菌并且在4℃。

致谢

该协议从Bigay和Antonny(2005)的工作进行了修改。这项研究得到了国家发展和改革委员会的资助。国民经济基督教基金会法国肌营养不良协会(AFM);以及国家中心科学院; RégionMidi-Pyrénées和欧洲基金(FEDER,FondsEuropéensdeDéveloppementRégional)。作者声明没有利益冲突或竞争利益。

参考文献

  1. Bigay,J. and Antonny,B.(2005)。  COP涂层的组装 - 拆解周期的实时测定在规定尺寸的脂质体上。方法Enzymol 404:95-107。
  2. Boal,F.,Mansour,R.,Gayral,M.,Saland,E.,Chicanne,G.,Xuereb,JM和Tronchère,H。(2015)。< a class ="ke-insertfile"href = "http://jcs.biologists.org/content/128/4/815.short"target ="_ blank"> TOM1是参与内体成熟调控的PI5P效应物。 128(4):815-827。
  3. Di Paolo,G。和De Camilli,P.(2006)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17035995"target =" _blank">磷酸肌醇在细胞调节和膜动力学中。自然 443(7112):651-657。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tronchere, H. and Boal, F. (2017). Liposome Flotation Assays for Phosphoinositide-protein Interaction. Bio-protocol 7(5): e2169. DOI: 10.21769/BioProtoc.2169.
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