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Extraction of Intracellular and Cell Wall Proteins from Leaves and Roots of Harsh Hakea
从粗糙哈克木属植物叶和根中提取胞内和细胞壁蛋白质

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Abstract

Plant proteins can be targeted to intracellular (i.e. cytosol, vacuole, organelles etc.) or extracellular (i.e., cell walls, apoplast) compartments. Dual targeting is a key mechanism with important implications for plant metabolism, growth, development and defense etc. Harsh Hakea (Hakea prostrata R.Br.) is a perennial species and member of the Proteaceae family that thrives on extremely phosphate impoverished soils of southwestern Australia. Harsh Hakea is not a common model organism, but has been widely developed for physiological and molecular/biochemical studies of the endogenous adaptations of an ‘extremophile’ plant species to abiotic stress, including low phosphorus tolerance. Tissues of Harsh Hakea contain large amounts of compounds (e.g., phenolics) that interfere with the extraction of soluble proteins. We previously optimised extraction of intracellular proteins from Harsh Hakea proteoid roots to improve soluble protein yield by at least 10-fold (Shane et al., 2013). Here, we describe the protocol for extraction and separation of intracellular from ‘loosely bound’ cell-wall proteins in Harsh Hakea.

Materials and Reagents

  1. 2 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-138 )
  2. Dialysis tubing (12-14 kDa MWCO) (Spectra/Por, Fisher Scientific, catalog number: 08-667B )
  3. 25 mm syringe filters (0.45 μm) (Sarstedt, catalog number: 83.1826 )
  4. Harsh Hakea leaves or roots
  5. Dewar (MVE 20/15) with liquid N2, and ice bucket
  6. Imidazole (Bioshop, catalog number: IMID508 )
  7. Triton X-100 (Bioshop, catalog number: TRX777 )
  8. Glycerol (Bioshop, catalog number: GLY002 )
  9. Magnesium chloride (Bioshop, catalog number: MAG508)
  10. Polyethylene glycol 8000 (Bioshop, catalog number: PEG800 )
  11. Phenylmethylsulfonyl fluoride (PMSF, G-Biosciences, catalog number: 786-0555 )
  12. Thiourea (Sigma, catalog number: T-7875 )
  13. Poly (vinylpolypyrrolidone) (PVPP, Sigma-Aldrich, catalog number: P-6755 )
  14. Poly (vinylpyrrolidone) (Bioshop, catalog number: PVP504 )
  15. Intracellular extraction buffer (see Recipes)
  16. Resuspension buffer (see Recipes)
  17. Cell wall extraction buffer (see Recipes)
  18. Dialysis buffer (see Recipes)

Equipment

  1. ZebaTM spin desalting columns 7K MWCO (0.5 ml) (Fisher Scientific, catalog number: 89882 )
  2. Porcelain mortar (70 mm inner diameter) and pestle
  3. Plastic pellet pestle (Kimble-Chase Kontes, Fisher Scientific, catalog number: K749521-0590 )
  4. Macrosep® Advance Centrifugal Concentrators (10 kDa MWCO) (Pall Corporation, catalog number: MAP010C37 )
  5. Microcentrifuge (Beckman Coulter, model: microfuge 22R )
  6. PT-3100 Polytron Homogenizer (Kinematica, model: PT-3100)
  7. Magnetic stirrer
  8. 4 °C cold room

Procedure

This protocol applies to extraction of intracellular and cell-wall proteins from Harsh Hakea leaves, but can also be applied to roots (e.g., Shane et al., 2014; Shane et al., 2013). For protocols optimized for extracting intracellular and cell wall proteins from leaves, stems etc. of the model plant Arabidopsis thaliana see Shane et al. (2014); Shane et al. (2013) and Robinson et al. (2012).

  1. Extraction of soluble intracellular proteins
    1. Quickly freeze freshly harvested tissues in liquid N2. Store at -80 °C until use.
    2. Grind frozen tissues (e.g., ~1 g, comprising a biological replicate) to a powder under liquid N2 using a pre-chilled mortar and pestle.
    3. Transfer an aliquot (e.g., ~200 mg) of frozen tissue powder to a pre-cooled, 2 ml microcentrifuge tube. Do not allow tissue to thaw during this step. Maintain samples in liquid N2 until all samples are weighed out.
    4. Add ice-cold Intracellular extraction buffer (IEB) at 1:4 (w/v) to each sample and vortex until tissue thaws into extraction buffer. Briefly homogenize sample using the Polytron (2 to 4 pulses at high speed for 10 to 15 sec per pulse) while maintaining the microcentrifuge tube on ice. Retain homogenates on ice until all extractions are completed (it is recommended not to exceed 8 to 10 samples per batch to reduce probability of protein proteolysis etc.).
    5. Centrifuge homogenates at 17,500 x g for 15 min at 4 °C.
    6. Carefully pipette off supernatant (clarified extract) and desalt using a ZebaTM spin desalting column previously equilibrated in IEB lacking PVPP and PVP. The desalted extract is designated the intracellular fraction of soluble proteins (e.g., ~2-10 mg protein/ml depending upon the tissue type and stage of development).

  2. Washing the insoluble pellets
    1. The pellet containing insoluble cell debris is washed twice by resuspending in 1.5-2.0 ml of re-suspension buffer (RB) using a pellet pestle, and centrifugation as above (supernatants are discarded).
    2. The pellet is washed as above an additional four times in RB lacking Triton X-100. When completely washed, the pellets will be nearly colourless.

  3. Extraction of cell-wall bound proteins
    1. The extraction of cell wall proteins from the washed insoluble pellets is done using a cell wall extraction buffer (CWEB) (see Recipes) (~ 2 ml) while stirring for 30 min at 4 °C.
    2. Samples are centrifuged as above and concentrated using the Macrosep® centrifugal concentrator to ~0.2 ml yielding a protein concentration of ~2 mg/ml [for spectrophotometric determination of protein concentration see Knowles and Plaxton (2013)].
    3. Clarify extracts by passing through the 0.45 syringe filter, and then dialyse overnight at 4 °C against 4 L of the dialysis buffer (DB) using Spectra/Por dialysis tubing (12-14 kDa MWCO, 25 mm flat width).
    4. The resultant sample contains ‘loosely-bound’ cell-wall proteins.
    5. SDS-PAGE of the cell wall versus intracellular fractions followed by immunoblotting using anti-aldolase and anti-phosphoenolpyruvate carboxylase antibodies is performed to confirm absence of immunoreactive -40 kDa aldolase and -100 kDa phosphoenolpyruvate carboxylase polypeptides (i.e., cytoplasmic marker proteins) in the cell wall extracts [for detailed procedure see Shane et al. (2014)].

Recipes

All buffers are made up fresh the day before and stored at 4 °C until used, but no sterilization was done.

  1. Intracellular extraction buffer (IEB, keep on ice)
    50 mM imidazole-HCl (pH 7.0)
    0.1% (v/v) Triton X-100
    10% (v/v) glycerol
    10 mM thiourea
    2 mM MgCl2
    2% (w/v) polyethylene glycol 8,000
    1% (w/v) polyvinyl (polypyrrolidone) (PVPP)
    Note: Add fresh to IEB immediately prior to tissue extraction.
    1% (w/v) polyvinylpyrrolidone (PVP)
    Note: Add fresh to IEB immediately prior to tissue extraction.
    1 mM phenylmethylsulphonyl fluoride (PMSF)
    Notes:
    1. Add fresh to IEB immediately prior to tissue extraction.
    2. Prepare a 100 mM stock of PMSF in absolute ethanol and store at -20 °C; PMSF is unstable in aqueous solution. Handle PMSF with care as this protease inhibitor is quite toxic.
  2. Resuspension buffer (RB, keep in ice)
    50 mM Tris-HCl (pH 7.4)
    10 mM MgCl2
    1% (v/v) Triton X-100
  3. Cell wall extraction buffer (CWEB, keep in ice)
    1 M NaCl in 40 mM Tris-HCl (pH 7.4)
    10 mM MgCl2
  4. Dialysis buffer (DB, keep in 4 °C cold room)
    40 mM Tris-HCl (pH 7.4)
    10 mM MgCl2

Acknowledgments

This work was supported by the Australian Research Council (grant no. DP1092856 to M. W. S.), as well as grants from the Natural Sciences and Engineering Research Council of Canada and Queen’s Research Chairs program (to W. C. P.).

References

  1. Knowles, V. and Plaxton, W. (2013). Protein extraction, acid phosphatase activity assays, and determination of soluble protein concentration. Bio-protocol 3(17): e889.
  2. Shane, M. W., Fedosejevs, E. T. and Plaxton, W. C. (2013). Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea. Plant Physiol 161(4): 1634-1644.
  3. Shane, M. W., Stigter, K., Fedosejevs, E. T. and Plaxton, W. C. (2014). Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae). J Exp Bot 65(20): 6097-6106.
  4. Robinson, W. D., Park, J., Tran, H. T., Del Vecchio, H. A., Ying, S., Zins, J. L., Patel, K., McKnight, T. D. and Plaxton, W. C. (2012). The secreted purple acid phosphatase isozymes AtPAP12 and AtPAP26 play a pivotal role in extracellular phosphate-scavenging by Arabidopsis thaliana. J Exp Bot 63(18): 6531-6542.

简介

植物蛋白可以靶向细胞内(即胞质溶胶,液泡,细胞器等)或细胞外(即细胞壁,质外体)区室。双重靶向是对植物新陈代谢,生长,发育和防御等具有重要影响的关键机制。 Harsh Hakea( Hakea prostrata R.Br.)是一种多年生物种和成员的泛革兰科家族,在澳大利亚西南部的极端磷酸盐贫困土壤上生长。 Harsh Hakea不是一种常见的模式生物,而是广泛开发用于"极端"植物物种对非生物胁迫(包括低磷耐性)的内源性适应的生理学和分子/生物化学研究。 Harsh Hakea的组织含有大量干扰可溶性蛋白质提取的化合物(例如,酚类化合物)。我们以前优化了来自Harsh Hakea蛋白质组织根的细胞内蛋白质的提取,将可溶性蛋白质产量提高至少10倍(Shane等人,2013)。在这里,我们描述的提取和细胞内分离从"松散绑定"细胞壁蛋白质在Harsh Hakea的协议。

材料和试剂

  1. 2ml微量离心管(Fisher Scientific,目录号:05-408-138)
  2. 透析管(12-14kDa MWCO)(Spectra/Por,Fisher Scientific,目录号:08-667B)
  3. 25mm注射器过滤器(0.45μm)(Sarstedt,目录号:83.1826)
  4. 苛刻的Hakea叶或根
  5. 具有液体N 2的杜瓦瓶(MVE 20/15)和冰桶
  6. 咪唑(Bioshop,目录号:IMID508)
  7. Triton X-100(Bioshop,目录号:TRX777)
  8. 甘油(Bioshop,目录号:GLY002)
  9. 氯化镁(Bioshop,目录号:MAG508)
  10. 聚乙二醇8000(Bioshop,目录号:PEG800)
  11. 苯甲基磺酰氟(PMSF,G-Biosciences,目录号:786-0555)
  12. 硫脲(Sigma,目录号:T-7875)
  13. 聚(乙烯基聚吡咯烷酮)(PVPP,Sigma-Aldrich,目录号:P-6755)
  14. 聚(乙烯基吡咯烷酮)(Bioshop,目录号:PVP504)
  15. 细胞内提取缓冲液(参见配方)
  16. 重悬缓冲液(见配方)
  17. 细胞壁提取缓冲液(参见配方)
  18. 透析缓冲液(参见配方)

设备

  1. Zeba TM旋转脱盐柱7K MWCO(0.5ml)(Fisher Scientific,目录号:89882)
  2. 瓷砂浆(内径70mm)和研钵
  3. 塑料颗粒杵(Kimble-Chase Kontes,Fisher Scientific,目录号:K749521-0590)
  4. Advance Centrifugal Concentrators(10kDa MWCO)(Pall Corporation,目录号:MAP010C37)
  5. 微量离心机(Beckman Coulter,型号:microfuge 22R)
  6. PT-3100 Polytron均化器(Kinematica,型号:PT-3100)
  7. 磁力搅拌器
  8. 4°C冷室

程序

该方案适用于从Harsh Hakea叶中提取细胞内和细胞壁蛋白,但也可以应用于根(例如,Shane等人,2014; Shane < em> et al。,2013)。对于优化用于从叶子,茎等提取细胞内和细胞壁蛋白的方案。拟南芥 参见Shane等人); Shane (2013)和Robinson 等人(2012)。

  1. 可溶性细胞内蛋白质的提取
    1. 快速冷冻新鲜收获的组织在液体N 2。储存于-80℃直至使用。
    2. 研磨冷冻组织(例如,?1g,包括生物学重复) ?使用预冷的研钵和研杵将其加入到液体N 2下的粉末中
    3. 将等分试样(例如,?200mg)的冷冻组织粉末转移到a 预冷,2ml微量离心管。不要让组织解冻 在此步骤。保持样品在液体N 2中直到所有样品 称重。
    4. 加入冰冷的细胞内提取缓冲液(IEB) 以1:4(w/v)的比例加入每个样品并涡旋直到组织融化 提取缓冲液。使用Polytron(2至4)简单均质化样品 脉冲高速,每脉冲10至15秒),同时保持 微量离心管在冰上。在冰上保留匀浆直到全部 提取完成(建议不超过8到10 样品/批次以减少蛋白质蛋白水解的可能性等)。
    5. 在4℃下以17,500×g离心匀浆15分钟
    6. 小心吸取上清(澄清的提取物)和脱盐 使用先前在IEB中平衡的Zeba TM 自旋脱盐柱 缺乏PVPP和PVP。脱盐的提取物称为 可溶性蛋白质的细胞内部分(例如?2-10mg蛋白质/ml) 取决于组织类型和发育阶段)。

  2. 洗涤不溶性小丸
    1. 含有不溶性细胞碎片的沉淀物被洗涤两次 使用沉淀重悬于1.5-2.0ml重悬浮缓冲液(RB)中 杵,和如上所述的离心(弃去上清液)
    2. 如上所述,将沉淀物在缺乏RB的条件下洗涤四次 Triton X-100。当完全洗涤时,粒料将近 无色。

  3. 细胞壁结合蛋白的提取
    1. 从洗涤的不溶性小丸中提取细胞壁蛋白 使用细胞壁提取缓冲液(CWEB)(参见Recipes)(?2 ml),同时在4℃下搅拌30分钟
    2. 将样品离心 ?并使用Macrosep离心浓缩器浓缩 ?0.2ml,产生?2mg/ml的蛋白质浓度[for 分光光度法测定蛋白质浓度见Knowles 和Plaxton(2013)]
    3. 通过0.45通过澄清提取物 ?注射器过滤器,然后在4℃下对4L透析过夜 透析缓冲液(DB),使用Spectra/Por透析管(12-14kDa MWCO, 25 mm平宽)。
    4. 所得样品含有"松散结合"的细胞壁蛋白。
    5. 细胞壁对细胞内部分的SDS-PAGE,随后 ?使用抗醛缩酶和抗磷酸烯醇丙酮酸的免疫印迹 羧化酶抗体以确认无免疫反应性 ?-40kDa醛缩酶和-100kDa磷酸烯醇丙酮酸羧化酶 多肽(即细胞质标记蛋白) 提取物[具体程序参见Shane等人(2014)]。

食谱

所有缓冲液在前一天新鲜配制并在4℃下储存直至使用,但不进行灭菌。

  1. 细胞内提取缓冲液(IEB,保存在冰上)
    50mM咪唑-HCl(pH7.0) 0.1%(v/v)Triton X-100 10%(v/v)甘油 10mM硫脲 2mM MgCl 2/
    2%(w/v)聚乙二醇8000 1%(w/v)聚乙烯基(聚吡咯烷酮)(PVPP) 注意:在提取组织前立即向IEB添加新鲜。
    1%(w/v)聚乙烯吡咯烷酮(PVP)
    注意:在提取组织前立即向IEB添加新鲜。
    1mM苯甲基磺酰氟(PMSF) 注意:
    1. 在提取组织之前,立即向IEB添加新鲜。
    2. 准备 ?100mM PMSF储备液,置于-20℃保存; PMSF是 在水溶液中不稳定。谨慎处理PMSF作为此蛋白酶 抑制剂相当毒。
  2. 重悬缓冲液(RB,保存在冰中)
    50mM Tris-HCl(pH7.4) 10mM MgCl 2/
    1%(v/v)Triton X-100
  3. 细胞壁提取缓冲液(CWEB,保存在冰中)
    1M NaCl的40mM Tris-HCl(pH7.4)中 10mM MgCl 2/
  4. 透析缓冲液(DB,保存在4℃冷室)
    40mM Tris-HCl(pH7.4) 10mM MgCl 2/

致谢

这项工作得到澳大利亚研究委员会(授予M.W.S.的授权号DP1092856)以及加拿大自然科学和工程研究理事会和皇后研究教席计划(给W.C.P.)的资助。

参考文献

  1. Knowles,V.和Plaxton,W。(2013)。 蛋白质提取,酸性磷酸酶活性测定以及可溶性蛋白质浓度的测定生物方案 3(17):e889。
  2. Shane,M. W.,Fedosejevs,E.T.和Plaxton,W.C。(2013)。 通过体内 单泛素化和磷酸化在发育中的相互作用控制anaplerotic phosphoenolpyruvate羧化酶磷酸盐缺乏的刺槐的蛋白质根。植物生理学 161(4):1634-1644。
  3. Shane,M. W.,Stigter,K.,Fedosejevs,E.T.和Plaxton,W.C。(2014)。 衰老诱导型细胞壁和细胞内紫色酸性磷酸酶:对于Hakea prostrata中的磷再移植的影响(Proteaceae)和 Arabidopsis thaliana (Brassicaceae)。 65(20):6097-6106。
  4. Robinson,W.D.,Park,J.,Tran,H.T.,Del Vecchio,H.A.,Ying,S.,Zins,J.L.,Patel,K.,McKnight,T.D.and Plaxton, 分泌的紫色酸性磷酸酶同功酶AtPAP12和AtPAP26在胞外磷酸盐清除中起着关键作用> Arabidopsis thaliana 63(18):6531-6542。
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引用:Shane, M. W. and Plaxton, W. (2015). Extraction of Intracellular and Cell Wall Proteins from Leaves and Roots of Harsh Hakea. Bio-protocol 5(23): e1678. DOI: 10.21769/BioProtoc.1678.
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