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Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp
从番茄果皮中分离液泡膜微囊   

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Abstract

This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.

Materials and Reagents

  1. Ultra centrifuge tubes, 32 ml capacity, thick wall, polycarbonate (Beckman Coulter, catalog number: 355631 )
  2. 3 ml pastettes
  3. Solanum lycopersicum Lam. cv. M82 or cv. Micro-Tom
  4. Bovine serum albumin (Fraction V) (Sigma-Aldrich, catalog number: A7906 )
  5. D-Mannitol (Sigma-Aldrich, catalog number: M9647 )
  6. Magnesium sulfate heptahydrate (Thermo Fisher Scientific, catalog number: 10256840 )
  7. Ethylenediaminetetraacetic acid disodium salt dihydrate (Sigma-Aldrich, catalog number: E4884 )
  8. Polyvinylpyrrolidone average molecular weight 40,000 (Sigma-Aldrich, catalog number: PVP40 )
  9. 2-Amino-2-(hydroxymethyl)-1, 3-propanediol (Trizma® base) (Sigma-Aldrich, catalog number: T1503 )
  10. Butylated hydroxytoluene (Sigma-Aldrich, catalog number: W218405 )
  11. Potassium disulfite (K2S2O5) (Sigma-Aldrich, catalog number: P2522 )
  12. Phenylmethylsulfonyl fluouride (Sigma-Aldrich, catalog number: P7626 )
  13. Ethanol (Thermo Fisher Scientific, catalog number: 12468750 )
  14. (+)-Sodium L-ascorbate (Sigma-Aldrich, catalog number: A7631 )
  15. DL-Dithiothreitol (Sigma-Aldrich, catalog number: D0632 )
  16. Potassium hydroxide (Thermo Fisher Scientific, catalog number: 10366240 )
  17. Glycerol (Thermo Fisher Scientific, catalog number: 10296200 )
  18. BIS-TRIS propane (Sigma-Aldrich, catalog number: B6755 )
  19. Tricine (Sigma-Aldrich, catalog number: T0377 )
  20. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  21. Muslin cloth (100 % cotton)
  22. 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: M3671 )
  23. Extraction buffer (see Recipes)
  24. Resuspension buffer (see Recipes)
  25. Sucrose steps (see Recipes)
  26. Transport buffer (see Recipes)

Equipment

  1. Scalpel Handle Swan Morton No.4 (Philip Harris, catalog number: B8R00181 )
  2. Scalpel blades Swan Morton No. 24 (Thermo Fisher Scientific, catalog number: 11712734 )
  3. Blender (For example a Waring 38BL41 with a 40 oz container)
  4. Ultra centrifuge capable of 100,000 x g with swing out rotor (e.g. SW28 Ti)
  5. Soft paint brush (Sable No.5 or equivalent)
  6. 1,000 µl micropipette

Procedure

The core procedure detailed here was designed to isolate high purity tonoplast vesicles for proteomic analysis. Modifications to the step gradient are included that allow higher yield preparations suitable for transport assays. As with all organelle isolation procedures the balance between yield and purity will depend on the downstream application. For example proteomic approaches are facilitated by a higher purity of membranes whilst transport assays require a higher yield of membrane. The scale of this protocol can be readily adapted for between 20 g to 80 g of starting material, maintaining the same proportion of plant biomass to buffer.
This procedure is carried out at 4 °C, in a cold room, using pre-chilled equipment and freshly prepared solutions incubated on ice for 4 h before starting.

  1. Homogenise 60 g of tomato fruit pericarp [exocarp (outer skin) removed] (see Figure 1) with 150 ml Extraction buffer (see Recipe 1) in a blender.
    Note: To minimise heating the fruit are homogenised in 6 x 3 sec high speed pulses.


    Figure 1. Preparation of tomato fruit pericarp for homogenisation. Panel A shows a quarter of a tomato fruit with the regions of pericarp tissue and the exocarp labelled. Panel B shows the same quarter prepared for homogenisation after removal of the exocarp and locule.

  2. Filter the homogenate through 2 layers of muslin and distribute evenly between 6 ultra-centrifuge tubes. Approximately 180 ml of filtrate is usually collected giving approximately 30 ml of filtrate per centrifuge tube.
    Note: The filtration should be done quickly and the transfer of foam should be avoided.
  3. Centrifuge the homogenate at 20,000 x g for 20 min.
    Note: This is an initial clarifying spin to remove cell debris and mitochondria.
  4. Transfer the supernatant to clean ultra-centrifuge tubes and centrifuge at 100,000 x g for 60 min.
    Note: This will pellet the membranes from the supernatant.
  5. During the centrifugation prepare 2 step gradients. In clean, dry ultra-centrifuge tubes layer 5 ml of 32% sucrose, 5 ml of 24% sucrose, 10 ml of 12% sucrose and 5 ml of 6% sucrose (see Recipe 3 for sucrose solutions). Keep the gradients at 4 °C until needed.
    Notes:
    1. If the membranes are intended for transport assays a simplified gradient consisting of 12 ml of 24% and 12 ml of 6% sucrose can be used.
    2. A schematic representation of the gradients is shown in Figure 2 A and C.
    3. A short video of how to layer sucrose solutions is shown in Video 1.

    Video 1. Layering sucrose solutions. The video shows the layering of 24% sucrose solution onto a layer of 32% sucrose. Note the starting position of the pastette just above the meniscus of the 32% sucrose layer. The pastette is raised slowly maintaining contact with the growing 24% layer.


    Figure 2. Sucrose step gradients for tonoplast vesicle isolation. Schematic representations of the sucrose step gradients prepared in step 5 for high purity, A, and high yield C protocols. Panels B and D show examples of the gradients after centrifugation in step 8. The interphases to be harvested are indicated with triangles. These gradients have been used to isolate membranes from mature red fruit.

  6. Remove the supernatant after centrifugation in step 4, ideally this is carried out using a 200 μl micro-pipette tip connected to an aspirator.
    Note: Keeping the end of the tip just below the surface of the supernatant will reduce the amount of liquid that sticks to the walls of the tube (you should hear a slurping noise when the tip is placed optimally). It is important to avoid touching the pellet with the tip as this will result in membrane loss.
  7. Resuspend all 6 pellets in a total of 5 ml of Resuspension buffer (see Recipe 2) using a soft paint brush. Gently sweep the tip of the brush across the membrane pellet until resuspended.
    Note: To maximise yields first resuspend the pellets in a total of 3 ml then rinse the tubes and brush with the remaining 2 ml and combine.
  8. Layer 2.5 ml of resuspended membranes onto the top of each gradient and centrifuge at 100,000 x g for 60 min.
  9. Harvest the membranes from the 6% to 12% interphase using a 1,000 µl pipette fitted with a tip cut at 45° (see Video 2) and transfer to a clean ultra-centrifuge tube.
    Notes:
    1. The membranes from the 2 gradients can be pooled at this point.
    2. Preps for transport assays should be harvested from the 6%-24% interphase.

    Video 2. Harvesting gradients. The video shows how to harvest an interphase from a sucrose step gradient. The initial removal of the upper phases above the desired interphase is carried out with a 3 ml pastette, this step reduces contamination from carry through as a pipette tip passes through a membrane layer. Care is taken not to disturb the target interphase leaving approximately 0.5 cm of solution above this layer. The membrane is harvested using a 1,000 µl pipette tip cut to 45◦.

  10. Add 20 ml of Resuspension buffer to the membranes and centrifuge at 100,000 x g for 60 min.
    Note: This will wash the membranes.
  11. Aspirate off the supernatant and resuspend the pellet in 200 µl of Resuspension buffer.
    Note: If the membranes are for use in transport assays resuspend in Transport buffer (see Recipe 4).
  12. For storage flash freeze membranes in liquid nitrogen and store at -80 °C.

Representative data


Figure 3. Assessment of vesicle purity and integrity. Membrane fraction purities from full A and simplified B gradients were assessed by the inhibition of ATPase activities by 500 μM sodium orthovanadate (inhibits plasma membrane ATPase), 500 μM sodium azide (inhibits mitochondrial ATPase), or 50 mM KNO3 (inhibits tonoplast ATPase). C. Tonoplast vesicle integrity from the 6 24% membrane fraction of the gradient in panel B was assessed by the increase in ATPase activity in the presence of the detergent Brij® 58 (final concentration 0.015 mg ml-1).

Notes

  1. Membrane fraction purity can be assessed through ATPase assays (Oleski et al., 1987) using specific inhibitors of tonoplast, mitochondrial and plasma membrane ATPase (Smith et al., 1984). Membrane H+-ATPase complexes are highly indicative of specific membranes; V-type, tonoplast; F-type, mitochondrial; P-type, plasma membrane. By assaying the rate of ATP hydrolysis in a membrane fraction and measuring the reduction in activity in the presence of specific inhibitors the relative purity of a membrane fraction can be determined, see Figure 3 for example.
  2. The intactness of membrane vesicles can be assessed through ATPase assays in the presence and absence of the non-ionic surfactant Brij® 58 (Sigma-Aldrich, catalog number: P5884).
  3. To remove peripheral and soluble proteins a carbonate wash of the membranes can be performed. This should not be done on vesicles required for transport assays as you will no longer have sealed vesicles.
  4. Membrane yield can be assessed through protein content using common protein concentration assays e.g., Bradford method.

Recipes

  1. Extraction buffer
    450 mM mannitol
    3 mM MgSO4.7H2O
    5 mM Na2-EDTA
    0.5% (w/v) PVP-40
    50 mM TRIS
    500 µM butylated hydroxyltoluene
    26 mM potassium disulfite
    200 mM sodium ascorbate
    0.5% (w/v) BSA
    1 mM PMSF
    10 mM DTT
    pH 8.0 (KOH)
  2. Resuspension buffer
    1.1 M Glycerol
    1 mM Na2-EDTA
    10 mM BTP
    2 mM DTT
    pH 8.0 (Tricine)
  3. Sucrose steps
    6, 12, 24 and 32% sucrose (w/v) in resuspension buffer
  4. Transport buffer
    50 mM 2-(N-morpholino)ethanesulfonic acid (MES)/BTP (pH 7.0)
    5 mM KCl
    5 mM MgSO4.7H2O
    1 mM DTT

Acknowledgments

The research leading to the development of this protocol was funded by the Biotechnology and Biological Sciences Research Council, UK (grant BB/H00338X/1) and Syngenta.

References

  1. Bettey, M. and Smith, J. A. (1993). Dicarboxylate transport at the vacuolar membrane of the CAM plant Kalanchoe daigremontiana: sensitivity to protein-modifying and sulphydryl reagents. Biochim Biophys Acta 1152(2): 270-279.
  2. Oleski, N., Mahdavi, P., Peiser, G. and Bennett, A. B. (1987). Transport properties of the tomato fruit tonoplast: I. Identification and characterization of an anion-sensitive H-ATPase. Plant Physiol 84(4): 993-996.
  3. Smith, J. A., Uribe, E. G., Ball, E., Heuer, S. and Luttge, U. (1984). Characterization of the vacuolar ATPase activity of the crassulacean-acid-metabolism plant Kalanchoe daigremontiana. Receptor modulating. Eur J Biochem 141(2): 415-420.
  4. Snowden, C. J., Thomas, B., Baxter, C. J., Smith, J. A. and Sweetlove, L. J. (2015). A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition. Plant J 81(5): 651-660.

简介

这个协议描述了从番茄果实隔离tonoplast囊泡。 使用该程序分离的囊泡具有足够高的纯度用于下游蛋白质组分析,同时保留用于功能测定的转运感受态。 该方法用于研究番茄果实成熟期间氨基酸的转运(Snowden等人,2015)并且基于Betty和Smith使用的程序(Bettey和Smith,1993)。 这样的囊泡可用于进一步研究代谢物在整个质膜上的动态转移,用于番茄果实发育期间的储存和代谢。

材料和试剂

  1. 超离心管,32ml容量,厚壁,聚碳酸酯(Beckman Coulter,目录号:355631)
  2. 3 ml pastettes
  3. Solanum lycopersicum Lam。简历。 M82或cv。微汤姆
  4. 牛血清白蛋白(级分V)(Sigma-Aldrich,目录号:A7906)
  5. D-甘露糖醇(Sigma-Aldrich,目录号:M9647)
  6. 硫酸镁七水合物(Thermo Fisher Scientific,目录号:10256840)
  7. 乙二胺四乙酸二钠盐二水合物(Sigma-Aldrich,目录号:E4884)
  8. 聚乙烯吡咯烷酮平均分子量为40,000(Sigma-Aldrich,目录号:PVP40)
  9. 2-氨基-2-(羟甲基)-1,3-丙二醇(Trizma底物)(Sigma-Aldrich,目录号:T1503)
  10. 丁基化羟基甲苯(Sigma-Aldrich,目录号:W218405)
  11. 将亚硫酸氢钾(K 2 S 2 O 5)(Sigma-Aldrich,目录号:P2522)
  12. 苯基甲基磺酰基fluouride(Sigma-Aldrich,目录号:P7626)
  13. 乙醇(Thermo Fisher Scientific,目录号:12468750)
  14. (+) - L-抗坏血酸钠(Sigma-Aldrich,目录号:A7631)
  15. DL-二硫苏糖醇(Sigma-Aldrich,目录号:D0632)
  16. 氢氧化钾(Thermo Fisher Scientific,目录号:10366240)
  17. 甘油(Thermo Fisher Scientific,目录号:10296200)
  18. BIS-TRIS丙烷(Sigma-Aldrich,目录号:B6755)
  19. Tricine(Sigma-Aldrich,目录号:T0377)
  20. 蔗糖(Sigma-Aldrich,目录号:SO389)
  21. 平纹布(100%纯棉)
  22. 2 - (N,N-吗啉代)乙磺酸(MES)(Sigma-Aldrich,目录号:M3671)
  23. 提取缓冲液(参见配方)
  24. 重悬缓冲液(见配方)
  25. 蔗糖步骤(参见食谱)
  26. 传输缓冲区(请参阅配方)

设备

  1. Scalpel Handle Swan Morton No.4(Philip Harris,目录号:B8R00181)
  2. 手术刀片Swan Morton No.24(Thermo Fisher Scientific,目录号:11712734)
  3. 搅拌机(例如带有40盎司容器的Waring 38BL41)
  4. 带有转出转子(例如SW28 Ti)的能力为100,000 x g的超离心机。
  5. 软漆刷(Sable No.5或同等品)
  6. 1,000μl微量移液器

程序

这里详述的核心程序设计为隔离高纯度质膜囊泡进行蛋白质组学分析。包括对梯度梯度的修饰,其允许适合于运输测定的更高产量的制备。与所有细胞器分离程序一样,产率和纯度之间的平衡将取决于下游应用。例如,蛋白质组学方法由更高纯度的膜促进,而转运测定需要更高的膜产量。该方案的规模可以容易地适用于20g至80g的起始材料,保持相同比例的植物生物质缓冲。
该程序在4℃,在冷室中,使用预冷设备和新制备的溶液在冰上孵育4小时,然后开始。

  1. 均质化在搅拌器中用150ml提取缓冲液(参见配方1)将60g番茄果实果皮[外皮(外皮)除去](参见图1)。
    注意:为了最小化加热,水果以6×3秒的高速脉冲匀浆。


    图1.用于均化的番茄果皮果皮的制备。图A显示了四分之一的番茄果实,其具有果皮组织和外果皮的区域标记。图B显示了在除去外果皮和荚果之后准备用于匀浆的同一季度
  2. 通过2层细棉布过滤匀浆,并均匀地分布在6个超离心管之间。通常收集约180ml滤液,每个离心管约有30ml滤液 注意:应该快速过滤,避免发泡。
  3. 将匀浆以20,000×g离心20分钟。
    注意:这是一个初始澄清旋转,以清除细胞碎片和线粒体。
  4. 将上清液转移至干净的超离心管中,并以100,000xg离心60分钟。
    注意:这将从上清液中沉淀膜。
  5. 在离心期间准备2步梯度。在干净,干燥的超离心管层中加入5ml 32%蔗糖,5ml 24%蔗糖,10ml 12%蔗糖和5ml 6%蔗糖(见蔗糖溶液的配方3)。保持梯度在4°C直到需要 注意:
    1. 如果膜用于转运测定,则简化梯度 ?由12ml的24%和12ml的6%蔗糖组成。
    2. 梯度的示意图如图2A和C所示。
    3. 视频1中显示了如何分层蔗糖溶液的简短视频。

    视频1.分层蔗糖溶液视频显示24%蔗糖溶液层叠在32% ?蔗糖。注意刚刚上方的pastette的起始位置 弯月面的32%蔗糖层。软膏慢慢升起 保持与增长的24%
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    图2.用于液泡膜囊泡分离的蔗糖步骤梯度在高纯度A和高产量C方案的步骤5中制备的蔗糖步骤梯度的示意图。图B和D显示了步骤8中离心后的梯度的实例。待收获的中间相用三角形表示。这些梯度已经用于从成熟的红色水果中分离膜
  6. 在步骤4中离心后除去上清液,理想地,这使用连接到吸气器的200μl微量移液管尖端进行。
    注意:将吸头的末端保持在上清液的表面以下会减少粘附在管壁上的液体量(当吸头最佳放置时,应该会听到嘶嘶声)。重要的是避免用尖端接触沉淀,因为这将导致膜损失。
  7. 使用软漆刷将所有6个丸重悬于总共5ml的重悬缓冲液(参见配方2)中。轻轻扫过刷的尖端穿过膜沉淀,直到重新悬浮 注意:为了最大化产量,首先将沉淀重悬在总共3ml中,然后冲洗管,并用剩余的2ml刷洗并合并。
  8. 将2.5ml重悬浮的膜铺在每个梯度的顶部并在100,000×g离心60分钟。
  9. 使用配备有45°尖端切割的1000μl移液管(参见视频2)从6%至12%的间期收获膜,并转移至干净的超离心管。
    注意:
    1. 在这一点上可以汇集来自2个梯度的膜。
    2. 用于转运测定的Preps应当从6%-24%的间期收获。

    视频2.收获渐变。 视频显示如何从蔗糖步骤收获中间相 梯度。初始除去高于期望的上部相 相间用3ml糊状物进行,这一步骤减少 当移液管尖端通过时,污染物从携带通过 膜层。注意不要扰乱目标相间 在该层上留下约0.5cm的溶液。膜 使用1000μl移液器尖端切割至45°收获。
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  10. 向膜中加入20ml的重悬缓冲液,并以100,000×g离心60分钟。
    注意:这会洗膜。
  11. 吸出上清液,并在200μl重悬缓冲液中重悬沉淀。
    注意:如果膜用于运输测定,则重新悬浮在运输缓冲液中(参见配方4)。
  12. 用于在液氮中储存闪速冷冻膜并储存于-80℃

代表数据


图3.囊泡纯度和完整性的评估。通过500μM原钒酸钠(抑制细胞膜ATP酶),500μM钠的抑制作用来评估来自完全A和简化B梯度的膜级分纯度叠氮化物(抑制线粒体ATP酶)或50mM KNO 3(抑制轮状体ATP酶)。 C.通过在洗涤剂Brij 58存在下ATP酶活性的增加来评估来自图B中梯度的6 24%膜级分的轮状体囊泡完整性(终浓度0.015mg ml -1 )。

笔记

  1. 膜级分纯度可以通过使用特定的原生质体,线粒体和质膜ATP酶的抑制剂(Smith等人,1984)通过ATP酶测定法(Oleski等人,1987)来评估。 。膜H sup + -ATPase复合物高度指示特异性膜; V型,tonoplast; F型,线粒体; P型,质膜。通过测定膜级分中ATP水解的速率并测量在特异性抑制剂存在下活性的降低,可以测定膜级分的相对纯度,例如参见图3。
  2. 膜囊泡的完整性可以通过在存在和不存在非离子表面活性剂58(Sigma-Aldrich,目录号:P5884)的情况下的ATP酶测定来评估。
  3. 为了除去外周和可溶性蛋白质,可以进行膜的碳酸盐洗涤。这不应该在运输检测所需的囊泡上进行,因为您将不再具有密封的囊泡
  4. 可以使用常见的蛋白质浓度测定法例如Bradford方法通过蛋白质含量评估膜产量。

食谱

  1. 提取缓冲
    450mM甘露糖 3mM MgSO 4。7H 2 O 3 5mM Na 2 EDTA-dTA 0.5%(w/v)PVP-40 50 mM TRIS
    500μM丁羟甲苯
    26 mM二硫酸钾 200mM抗坏血酸钠
    0.5%(w/v)BSA 1mM PMSF
    10 mM DTT
    pH 8.0(KOH)
  2. 悬浮缓冲液
    1.1 M甘油
    1mM Na 2 EDTA-EDTA 10 mM BTP
    2 mM DTT
    pH 8.0(Tricine)
  3. 蔗糖步骤
    6,12,24和32%蔗糖(w/v)的重悬缓冲液中
  4. 传输缓冲区
    50mM 2-(N-吗啉代)乙磺酸(MES)/BTP(pH 7.0)。
    5 mM KCl
    5mM MgSO 4。7H 2 O 3 1 mM DTT

致谢

导致该协议的研究的研究由英国生物技术和生物科学研究委员会(授权BB/H00338X/1)和先正达资助。

参考文献

  1. Bettey,M。和Smith,J.A。(1993)。 CAM植物Kalanchoe daigremontiana的液泡膜上的二羧酸盐转运:对蛋白修饰和巯基试剂的敏感性。 Biochim Biophys Acta 1152(2):270-279。
  2. Oleski,N.,Mahdavi,P.,Peiser,G。和Bennett,A.B。(1987)。 番茄果实叶绿体的转运性质:I.阴离子敏感性H-ATP酶的鉴定和表征。 Plant Physiol 84(4):993-996。
  3. Smith,J.A.,Uribe,E.G.,Ball,E.,Heuer,S。和Luttge,U。(1984)。 水稻腺苷酸代谢植物Kalanchoe daigremontiana的液泡ATPase活性的表征。受体调节。 Eur J Biochem 141(2):415-420。
  4. Snowden,C.J.,Thomas,B.,Baxter,C.J.,Smith,J.A。和Sweetlove,L.J。(2015)。 影响番茄果实氨基酸组成的tonoplast Glu/Asp/GABA交换剂。 em> Plant J 81(5):651-660。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Snowden, C. J., Thomas, B., Baxter, C. J., Smith, J. C. and Sweetlove, L. J. (2015). Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp. Bio-protocol 5(24): e1686. DOI: 10.21769/BioProtoc.1686.
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