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In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves
对苦草属叶片微粒体和浆膜切片中的潜在蓝光受体向光素进行体外磷酸化测定   

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Journal of Integrative Plant Biology
Jan 2015

Abstract

An aquatic angiosperm Vallisneria (Alismatales: Hydrocharitaceae) has been used as an excellent experimental material over a century to study the light regulation of dynamic intracellular movements including chloroplast redistribution and cytoplasmic streaming (Senn, 1908; Seitz, 1987; Takagi, 1997). However, understanding of the molecular mechanisms lagged behind because of difficulty in applying modern techniques such as gene transformation to this plant. Especially, which kind of photoreceptors function in these intriguing responses has long been an unsolved topic. Recently, genes encoding plant-specific blue-light receptor phototropins were isolated in Vallisneria, for the first time from aquatic plants (Sakai et al., 2015). Phototropins were identified first as the photoreceptor for hypocotyl phototropism in Arabidopsis thaliana, and now known to regulate many responses including chloroplast photorelocation movements in various plant species (Christie, 2007). Phototropins are localized mainly on the plasma membrane and their auto-phosphorylation induced by blue light is the critical step of signal transduction pathway (Sakamoto and Briggs, 2002; Kong et al., 2006; Kong et al., 2013; Inoue et al., 2010). Here we describe a protocol for in vitro protein phosphorylation assay using crude-microsomal and plasma-membrane-enriched fractions of Vallisneria, which enabled us to verify the presence of phototropins and characterize their auto-phosphorylation responses. After these analyses, Sakai et al. (2015) proposed that Vallisneria phototropins mediate the high-intensity-blue-light-induced chloroplast avoidance response.

Keywords: Crude microsomal fraction (粗微粒体级分), Plasma-membrane-enriched fraction (血浆膜富集级分), Light-induced phosphorylation (光诱导的磷酸化), Aquatic plants (水生植物), SDS-PAGE (SDS-PAGE)

Materials and Reagents

  1. Commonly-used medical cotton gauze
  2. 0.2 ml tube
  3. Vallisneria plants
    Note: Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured to be grown over 100 cm in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1A). The plants were grown under 12 h light/12 h dark regime at 20-24 °C.
  4. Dextran (from Leuconostoc ssp., Mr ~500,000) (Fluka, catalog number: 31392 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 31392 ”.
  5. Polyethylene glycol (Sigma-Aldrich, catalog number: P-3640 )
  6. SDS-PAGE
  7. Deionized water
  8. 1% Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
  9. 50 mM MOPS-KOH (pH7.6) (DOJINDO, catalog number: 343-01805 )
  10. 300 mM sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 196-00015 )
  11. 10 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) (DOJINDO, catalog number: 342-01314 )
  12. 5 mM ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
  13. 10 μg/ml dibutylhydroxytoluene (Tokyo Chemical Industry, catalog number: D0228 )
  14. 1% casein (Nacalai tesque, catalog number: 073-19 )
  15. 5 mM K2S2O5 (Wako Pure Chemical Industries, Siyaku, catalog number: 161-03345 )
  16. 1 mM dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 048-29224 )
  17. 2.5 mg/ml pepstatin A (Sigma-Aldrich, catalog number: P-4265 )
  18. 2.5 mg/ml aprotinin (Sigma-Aldrich, catalog number: A-4529 )
  19. 20 mg/ml EDTA-washed Polyvinylpolypyrolidone (Sigma-Aldrich, catalog number: P-6755 )
  20. MOPS-KOH (DOJINDO, catalog number: 345-02225 )
  21. 25 mM MgCl2 (Wako Pure Chemical Industries, Siyaku, catalog number: 135-00165 )
  22. 250 mM KCl (Wako Pure Chemical Industries, Siyaku, catalog number: 163-03545 )
  23. 40% glycerol (Wako Pure Chemical Industries, Siyaku, catalog number: 075-00616 )
  24. 20% 2-mercaptoethanol (Sigma-Aldrich, catalog number: A-6365 )
  25. A small amount of bromophenol blue (Wako Pure Chemical Industries, Siyaku, catalog number: 021-02911 )
  26. 0.5 M Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: 252859 )
  27. Homogenizing medium (see Recipes)
  28. Buffer A (see Recipes)
  29. Buffer B (see Recipes)
  30. 5x phosphorylation buffer (see Recipes)
  31. ATP mixture (see Recipes)
  32. 4x SDS buffer (see Recipes)

Equipment

  1. Blade (Lion Office Products Corp., model: L-300 )
  2. Polytron homogenizer (Kinematica AG, model: PT-35/ 2ST”OD” )
  3. Centrifuge
  4. Ultracentrifuge
  5. Teflon homogenizer (10 ml) (Ikemoto Scientific Technology, model: 812-771-04 )
  6. Lighting system (Sugiura Lab, model: FI-150T )
  7. Cut-off filter (KenkoTokina Corporation, model: Y-44 )
  8. Interference filter (KenkoTokina Corporation, model: BP-45 )
  9. Autoradiography (Fujifilm Corporation)

Procedure

  1. Plant material and pretreatment of specimens
    Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured to be grown over 100 cm in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1A). The plants were grown under 12 h light/12 h dark regime at 20-24 °C. The light source was a bank of 20 W fluorescent lamps (50 μmol m-2 s-1).

  2. Preparation of crude microsomal (CM) fraction and plasma membrane-enriched (PM) fraction
    1. At the end of the light period, 40 g of the healthy fresh leaves were harvested into tap water and kept in complete darkness for 12-16 h.
      Note: Before use, snip off unhealthy part from the leaves.
    2. The leaves were chopped on ice with a blade (Figure 1B). These small leaf pieces were homogenized with a Polytron homogenizer (Figure 1C) in 120 ml of a homogenizing medium.
    3. The homogenate was filtrated through six sheets of gauze (Figure 1D) and centrifuged (8,000 x g, 15 min, 4 °C).
    4. The supernatant (Figure 1E) was ultracentrifuged at 156,000 x g for 1 h at 4 °C.
    5. The pellet was suspended with a Teflon homogenizer in Buffer B (Figure 1F), followed by another ultracentrifugation at 156,000 x g for 1 h at 4 °C.
    6. The pellet (Figure 1G) was washed with buffer A and then re-suspended into a small volume (usually 6 ml) of the fresh buffer A, designated as a CM fraction. When the CM fraction was directly used for phosphorylation assay, it was washed and suspended with buffer B, not buffer A.
    7. A highly purified PM fraction was isolated from the CM fraction by applying aqueous polymer two-phase partitioning (Figure 1H-I) using Dextran from Leuconostoc ssp. Mr ~500,000 and polyethylene glycol (Yoshida et al., 1983; Takagi et al., 1988; Harada et al., 2002).
    8. After the final ultracentrifugation of the polyethylene-glycol-enriched upper phase, the pellet was washed with buffer B and then re-suspended into a small volume of the fresh buffer, designated as a PM fraction.
      Note: All the procedures were carried out on ice at 0-4 °C under dim red light (0.1 μmol m-2 s-1). The total protein contents in the CM and PM fractions were determined by Bradford assay. The CM and PM fractions were kept in darkness on ice until use.


    Figure 1. Preparation steps of CM and PM fractions. A. Vallisneria plants cultured in buckets. B. Leaves were chopped on ice. C. Leaves were further homogenized with a Polytron homogenizer. D. The homogenate was filtrated through six sheets of gauze. E. The filtrated homogenate before low-speed centrifugation (8,000 x g, 15 min) to remove debris such as cell wall. F. The pellet after the first ultracentrifugation (156,000 x g, 1 h) was suspended with a Teflon homogenizer. G. The pellet after the second ultracentrifugation (156,000 x g, 1 h). H and I. The plasma-membrane-enriched upper phase was separated from the lower phase after the first (H) and second (I) two-phase partitioning.

  3. In vitro phosphorylation assay
    1. The CM and PM fraction were suspended in the reaction mixture as described below.
      Reaction mixture (17.5 μl for each reaction)
      CM and PM fraction containing 20 μg protein   
      x μl
      5x phosphorylation buffer                               
      4 μl
      1% Triton X-100                                            
      1 μl
      Deionized water                                             
      17.5-x μl
    2. The reaction mixture put into a 0.2 ml tube was irradiated with blue light (450 nm) for a defined period of time (usually 0-300 sec) using a lighting system equipped with a fiber scope, a cut off filter, and an interference filter (Figure 2A).
    3. Immediately after the end of the irradiation, the reaction mixture was mixed with ATP mixture (2.5 μl) containing radioactive ATP and incubated for 2 min to allow phosphorylation reaction. The reaction was terminated by the addition of 4x SDS buffer (7 μl).
      Note: Steps C1-3 were carried out on ice under dim red light (0.1 μmol m-2 s-1) in the dark room to avoid any non-specific excitation of photoreceptors by background light.
    4.  The sample was boiled for 2 min and subjected to SDS-PAGE in a 10.0% (w/v) gel. Separated polypeptides on the gel were stained once with Coomassie Brilliant Blue to visualize the protein profiles (Figure 2B, the left panel).
      Note: In each lane, 20 μg of proteins were loaded.
    5. Autoradiography was carried out by exposing the de-stained and dried-up gels to an imaging plate for 0.5-1 h at room temperature (Figure 2B, the right panel).


      Figure 2. Irradiation and autoradiography. A. Irradiation of a sample in a 0.2-ml tube on ice using a lighting system equipped with a fiber scope, a cut-off filter and an interference filter. B. A dried-up gel stained with Coomassie Brilliant Blue (CBB; the left panel) and its autoradiograph (the right panel). Before (D) and after (L) the actinic irradiation, the protein-phosphorylation levels were examined in a crude microsomal fraction (CM) and a plasma-membrane-enriched fraction (PM). M; molecular size markers, arrowhead; radioactive signal of phosphorylation of Vallisneria phototropins.

Recipes

  1. Homogenizing medium
    50 mM MOPS-KOH (pH 7.6)
    300 mM sucrose
    10 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA)
    5 mM ethylenediaminetetraacetic acid (EDTA)
    10 μg/ml dibutylhydroxytoluene
    1% casein
    5 mM K2S2O5
    1 mM dithiothreitol (DTT)
    2.5 mg/ml pepstatin A
    2.5 mg/ml aprotinin
    20 mg/ml EDTA-washed Polyvinylpolypyrolidone
    Note: Contents of the medium were mixed with a magnetic stirrer at 4 °C overnight (12-16 h), except K2S2O5, DTT, pepstatin A, aprotinin, and EDTA-washed Polyvinylpolypyrolidone, which were added immediately before use. Polyvinylpolypyrolidone was previously washed with 5-mM EDTA, filtrated with a filter paper and dried up on the filter paper at room temperature for 1-2 days.
  2. Buffer A
    10 mM K-phosphate (pH 7.8)
    250 mM sucrose
  3. Buffer B
    5 mM MOPS-KOH (pH7.6)
    250 mM sucrose
    0.1 mM DTT
  4. 5x phosphorylation buffer
    250 mM Mops-KOH (pH 7.0)
    25 mM MgCl2
    250 mM KCl
    5 mM DTT
  5. ATP mixture (2.5 μl for each reaction)
    10 mCi/ml [γ-32P] ATP 0.5 μl
    20 μM ATP 2 μl
  6. 4x SDS buffer
    8% sodium dodecyl sulfate (SDS)
    40% glycerol
    20% 2-merchaptethanol
    A small amount of bromophenol blue
    0.5 M Tris-HCl (pH 6.8)

Acknowledgments

This protocol was modified from previous work by Takagi et al. (1988) and Harada et al. (2002). We thank Mr. Motoyuki Iida for taking pictures of culture facilities of Vallisneria plants (Figure 1A).

References

  1. Christie, J. M. (2007). Phototropin blue-light receptors. Annu Rev Plant Biol 58: 21-45.
  2. Harada, A., Okazaki, Y. and Takagi, S. (2002). Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. I. Regulation of activity during light-induced membrane hyperpolarization. Planta 214(6): 863-869.
  3. Inoue, S., Takemiya, A. and Shimazaki, K. (2010). Phototropin signaling and stomatal opening as a model case. Curr Opin Plant Biol 13(5): 587-593.
  4. Kong, S. G., Suzuki, T., Tamura, K., Mochizuki, N., Hara-Nishimura, I. and Nagatani, A. (2006). Blue light-induced association of phototropin 2 with the Golgi apparatus. Plant J 45(6): 994-1005.
  5. Kong, S. G., Suetsugu, N., Kikuchi, S., Nakai, M., Nagatani, A. and Wada, M. (2013). Both phototropin 1 and 2 localize on the chloroplast outer membrane with distinct localization activity. Plant Cell Physiol 54(1): 80-92.
  6. Sakai, Y., Inoue, S., Harada, A., Shimazaki, K. and Takagi, S. (2015). Blue-light-induced rapid chloroplast de-anchoring in Vallisneria epidermal cells. J Integr Plant Biol 57(1): 93-105.
  7. Sakamoto, K. and Briggs, W. R. (2002). Cellular and subcellular localization of phototropin 1. Plant Cell 14(8): 1723-1735.
  8. Seitz K (1987) Light-dependent movement of chloroplsts in higher plant cell. ACTA PHYSIOLOGIAE PLANTARUM 9: 137-148
  9. Senn, G. (1908). Die Gestalts- und Lageveränderung der Pflanzen-Chromatophoren. Verlag von Wilhelm Engelmann, Leipzig (in Germany).
  10. Takagi, S., Yoshida, S. and Nagai, R. (1988). A Model for Cell Membrane Isoration Using Vallisneria Leaves. Methods in bryology. Proc Bryol Meth, 81-88.
  11. Takagi, S. (1997). Photoregulation of cytoplasmic streaming: cell biological dissection of signal transduction pathway. J Plant Res. 110: 299-303.
  12. Yoshida, S., Uemura, M., Niki, T., Sakai, A. and Gusta, L. V. (1983). Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants. Plant Physiol 72(1): 105-114.

简介

水生被子植物Vallisneria(Alismatales:Hydrocharitaceae)已经在一个世纪内用作研究动态细胞内运动的光调节的优良实验材料,包括叶绿体再分布和细胞质流(Senn,1908; Seitz,1987 ; Takagi,1997)。然而,对分子机制的理解落后,因为难以应用现代技术,如基因转化到这个植物。特别地,哪种类型的光感受器在这些有趣的反应中起作用长期以来一直是未解决的主题。最近,在水生植物中首次在Vallisneria中分离编码植物特异性蓝光受体光托品的基因(Sakai等人,2015)。光金蛋白首先被鉴定为拟南芥中下胚轴向往性的光感受器,并且现在已知调节许多反应,包括各种植物物种中的叶绿体光定位运动(Christie,2007)。光催化素主要位于质膜上,并且它们由蓝光诱导的自磷酸化是信号转导途径的关键步骤(Sakamoto和Briggs,2002; Kong等,2006; et al。,2013; Inoue et al。,2010)。在这里,我们描述了使用Vallisneria的原始微粒体和质膜富集级分的体外蛋白磷酸化测定的方案,其使得我们能够验证光催化素的存在并表征它们的自身磷酸化反应。在这些分析之后,Sakai等人(2015)提出 Vallisneria 光催化素介导高强度蓝光诱导的叶绿体回避反应。

关键字:粗微粒体级分, 血浆膜富集级分, 光诱导的磷酸化, 水生植物, SDS-PAGE

材料和试剂

  1. 常用的医用棉纱布
  2. 0.2 ml管
  3. 注意:在热带鱼商店购买20-30cm长的Vallisneria的幼苗,并培养以在填充有自来水和底部的一层土壤的桶(20L)中生长超过100cm (图1A)。植物在12-24小时光照/12小时黑暗条件下在20-24℃生长。
  4. 葡聚糖(来自Leuconostoc ssp。,Mr?500,000)(Fluka,目录号:31392)
    注意:目前,它是"Sigma-Aldrich,目录号:31392"。
  5. 聚乙二醇(Sigma-Aldrich,目录号:P-3640)
  6. SDS- PAGE
  7. 去离子水
  8. 1%Triton X-100(Sigma-Aldrich,目录号:T-9284)
  9. 50mM MOPS-KOH(pH7.6)(DOJINDO,目录号:343-01805)
  10. 300mM蔗糖(Wako Pure Chemical Industries,Siyaku,目录号:196-00015)
  11. 10mM乙二醇双(2-氨基乙基醚)-N,N,N',N', - 四乙酸(EGTA)(DOJINDO,目录号:342-01314)
  12. 5mM乙二胺四乙酸(EDTA)(DOJINDO,目录号:345-01865)
  13. 10μg/ml二丁基羟基甲苯(Tokyo Chemical Industry,目录号:D0228)
  14. 1%酪蛋白(Nacalai tesque,目录号:073-19)
  15. 5mM K 2 SS 2 O 5(Wako Pure Chemical Industries,Siyaku,目录号:161-03345)。< br /
  16. 1mM二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:048-29224)
  17. 2.5mg/ml胃蛋白酶抑制剂A(Sigma-Aldrich,目录号:P-4265)
  18. 2.5mg/ml抑肽酶(Sigma-Aldrich,目录号:A-4529)
  19. 20mg/ml EDTA洗涤的聚乙烯聚吡咯烷酮(Sigma-Aldrich,目录号:P-6755)
  20. MOPS-KOH(DOJINDO,目录号:345-02225)
  21. 25mM MgCl 2(Wako Pure Chemical Industries,Siyaku,目录号:135-00165)
  22. 250mM KCl(Wako Pure Chemical Industries,Siyaku,目录号:163-03545)
  23. 40%甘油(Wako Pure Chemical Industries,Siyaku,目录号:075-00616)
  24. 20%2-巯基乙醇(Sigma-Aldrich,目录号:A-6365)
  25. 少量溴酚蓝(Wako Pure Chemical Industries,Siyaku,目录号:021-02911)
  26. 0.5M Tris-HCl(pH6.8)(Sigma-Aldrich,目录号:252859)
  27. 均质介质(见配方)
  28. 缓冲区A(参见配方)
  29. 缓冲液B(参见配方)
  30. 5x磷酸化缓冲液(参见配方)
  31. ATP混合物(参见配方)
  32. 4x SDS缓冲液(参见配方)

设备

  1. Blade(Lion Office Products Corp.,型号:L-300)
  2. Polytron匀浆器(Kinematica AG,型号:PT-35/2ST"OD")
  3. 离心机
  4. 超速离心机
  5. Teflon匀浆器(10ml)(Ikemoto Scientific Technology,型号:812fect04)
  6. 照明系统(Sugiura Lab,型号:FI-150T)
  7. 截止滤光器(Kenko Tokina Corporation,型号:Y-44)
  8. 干涉滤光器(Kenko Tokina Corporation,型号:BP-45)
  9. 放射自显影(富士胶片公司)

程序

  1. 植物材料和标本的预处理
    在热带鱼店购买长度为20-30cm的长春花属的年轻植物,并培养以在盛有自来水的桶(20L)中生长超过100cm,并且在其上铺有一层土壤底部(图1A)。植物在12-24小时光照/12小时黑暗条件下在20-24℃生长。光源是一组20W荧光灯(50μmolm-2s -1 )。

  2. 制备粗微粒体(CM)级分和富含质膜的(PM)级分
    1. 在光照期结束时,40g的健康新鲜叶 收获到自来水中并保持在完全黑暗中12-16小时。
      注意:使用前,从叶子上剪掉不健康的部分。
    2. 用刀片将叶子在冰上切碎(图1B)。这些小 叶片用Polytron匀浆器(图1C)匀浆 120ml匀浆培养基。
    3. 将匀浆通过六片纱布(图1D)过滤并离心(8,000×g,15分钟,4℃)。
    4. 将上清液(图1E)在156,000×g下在4℃超速离心1小时。
    5. 用Teflon匀浆器将沉淀悬浮在缓冲液B中(图1) 1F),然后在4℃下以156,000xg再次超速离心1小时 ?C。
    6. 用缓冲液A洗涤沉淀(图1G),然后 重悬于小体积(通常6ml)的新鲜缓冲液A中, 指定为CM分数。当直接使用CM部分时 磷酸化测定,洗涤并用缓冲液B悬浮,而不是 缓冲区A.
    7. 从CM中分离出高度纯化的PM级分 通过应用水性聚合物两相分配(图 1H-1)使用来自明串珠菌(Leuconostoc ssp。约500,000和聚乙烯 乙二醇(Yoshida等人,1983; Takagi等人,1988; Harada等人,2002)。
    8. 最后超速离心后 富含聚乙二醇的上层相,将沉淀物洗涤 缓冲液B,然后重新悬浮在小体积的新鲜缓冲液中, 指定为PM分数 注意:所有的程序都进行 在0-4℃下在暗红光(0.1μmolm -2 s -1 s -1 -1)下在冰上进行。总数 通过Bradford测定CM和PM级分中的蛋白质含量 测定。将CM和PM级分在冰上保存在黑暗中直到使用。


    图1. CM和PM级分的制备步骤。 Vallisneria B.将叶子在冰上切碎。 C.将叶子用Polytron匀浆器进一步匀浆。 D.将匀浆通过六片纱布过滤。 E.在低速离心(8,000×g,15分钟)之前过滤匀浆以除去碎屑,??例如细胞壁。 F.用Teflon匀浆器将第一次超速离心(156,000×g,1小时)后的沉淀物悬浮。 G.第二次超速离心后的沉淀(156,000×g,1小时)。 H和I.在第一(H)和第二(I)两相分配后,将富含质膜的上层相与下层相分离。

  3. 体外磷酸化测定
    1. 如下所述将CM和PM部分悬浮在反应混合物中 反应混合物(每个反应17.5μl)
      CM和PM级分含有20μg蛋白质 
      x微博
      5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液5x磷酸化缓冲液       
      4微升
      1%Triton X-100                     ;                    
      1微升
      去离子水                        ;                    
      17.5-x微克
    2. 将放入0.2ml管中的反应混合物用蓝色照射 光(450nm)照射规定的时间(通常为0-300秒),使用a 照明系统,配有光纤适配器,截止滤波器和 干扰滤波器(图2A)。
    3. 紧接着结束后 ?照射,将反应混合物与ATP混合物(2.5μl) 并孵育2分钟以允许 磷酸化反应。通过加入终止反应 ?4×SDS缓冲液(7μl) 注意:步骤C1-3在冰下进行 ?暗红光(0.1μmolm-2 s-1)在黑暗的房间,以避免任何 通过背景光的非特异性激发光感受器。
    4. 将样品煮沸2分钟,并在10.0%SDS中进行SDS-PAGE, (w/v)凝胶。凝胶上的分离的多肽被染色一次 考马斯亮蓝可视化蛋白质谱(图2B, 左面板)。
      注意:在每个泳道中,加载了20μg蛋白质。
    5. 通过暴露脱色和干燥进行放射自显影 ?凝胶到成像板在室温下0.5-1小时(图2B, 右面板)。


      图2.照射和放射自显影。 A.使用照明在冰上在0.2ml管中照射样品 系统配有光纤适配器,截止滤波器和干扰 ?过滤。 B.用考马斯亮蓝染色的干燥凝胶(CBB; 左图)和其放射自显影图(右图)。之前(D)和 ?在(L)光化辐照后,蛋白质磷酸化水平 在粗微粒体级分(CM)和a 质膜富集级分(PM)。 M;分子大小标记, 箭头; Vallisneria的光致磷酸化的放射性信号。

食谱

  1. 均质介质
    50mM MOPS-KOH(pH7.6) 300mM蔗糖 10mM乙二醇双(2-氨基乙基醚)-N,N,N',N', - 四乙酸(EGTA)
    5mM乙二胺四乙酸(EDTA) 10μg/ml二丁基羟基甲苯 1%酪蛋白
    5mM K 2 subS 2 O 5 sub/2 1mM二硫苏糖醇(DTT) 2.5mg/ml胃酶抑素A
    2.5mg/ml抑肽酶
    20mg/ml EDTA洗涤的聚乙烯吡咯烷酮 注意:使用磁力搅拌器将介质的内容物在4℃下混合过夜(12-16小时),除了K 2 subS 2 O 2外, 5,DTT,胃蛋白酶抑制剂A,抑肽酶和EDTA洗涤的聚乙烯基吡咯烷酮,其在使用前立即加入。预先用5-mM EDTA洗涤聚乙烯吡咯烷酮,用滤纸过滤,并在滤纸上在室温下干燥1-2天。
  2. 缓冲区A
    10mM K-磷酸(pH7.8) 250mM蔗糖
  3. 缓冲区B
    5mM MOPS-KOH(pH7.6) 250mM蔗糖 0.1 mM DTT
  4. 5x磷酸化缓冲液
    250mM Mops-KOH(pH7.0) 25mM MgCl 2·h/v 250 mM KCl
    5 mM DTT
  5. ATP混合物(每个反应2.5μl) 10mCi/ml [μ-γ-32P] ATP0.5μl
    20μMATP2μl
  6. 4x SDS缓冲液
    8%十二烷基硫酸钠(SDS)
    40%甘油 20%的2-巯基乙醇 少量溴酚蓝
    0.5M Tris-HCl(pH 6.8)

致谢

该协议从Takagi等人(1988)和Harada等人(2002)的先前工作修改。我们感谢Motoyuki Iida先生拍摄了Vallisneria植物的培养设施(图1A)。

参考文献

  1. Christie,J.M。(2007)。 Phototropin蓝光受体 Annu Rev植物生物 58:21-45。
  2. Harada,A.,Okazaki,Y。和Takagi,S。(2002)。 Vallisneria叶片中细胞膜H + -ATPase的光合控制。 I.在光诱导的膜超极化期间的活性调节。 214(6):863-869。
  3. Inoue,S.,Takemiya,A。和Shimazaki,K。(2010)。 光催化信号传导和气孔开放作为模型案例。Curr Opin Plant Biol 13(5):587-593。
  4. Kong,S.G.,Suzuki,T.,Tamura,K.,Mochizuki,N.,Hara-Nishimura,I.and Nagatani,A。(2006)。 蓝光诱导的phototropin 2与高尔基体的结合。植物J 45(6):994-1005。
  5. Kong,S.G.,Suetsugu,N.,Kikuchi,S.,Nakai,M.,Nagatani,A.and Wada,M.(2013)。 phototropin 1和2都位于叶绿体外膜上,具有不同的定位活性。 em> Plant Cell Physiol 54(1):80-92。
  6. Sakai,Y.,Inoue,S.,Harada,A.,Shimazaki,K.and Takagi,S。(2015)。 蓝光诱导的Vallisneria中的快速叶绿体解旋表皮细胞。 Integral Plant Biol 57(1):93-105。
  7. Sakamoto,K。和Briggs,W.R。(2002)。 光托品1的细胞和亚细胞定位。 植物细胞 14(8):1723-1735。
  8. Seitz K(1987)Light-dependent movement of chloroplsts in higher plant cell。 ACTA PHYSIOLOGIAE PLANTARUM 9:137-148
  9. Senn,G.(1908)。 Die Gestalts- undLagever?nderungder Pflanzen-Chromatophoren。 (位于德国)。
  10. Takagi,S.,Yoshida,S。和Nagai,R。(1988)。使用Vallisneria叶的细胞膜成孔的模型。方法在bryology。 Proc Bryol Meth ,81-88。
  11. Takagi,S。(1997)。 细胞质流的光调节:信号转导途径的细胞生物学分离。植物研究。 110:299-303。
  12. Yoshida,S.,Uemura,M.,Niki,T.,Sakai,A.and Gusta,L.V。(1983)。 在水性双聚合物相系统中分离膜颗粒及其实际用于纯化质膜的方法植物。植物生理学 72(1):105-114。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sakai, Y., Inoue, S. and Takagi, S. (2015). In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves. Bio-protocol 5(21): e1647. DOI: 10.21769/BioProtoc.1647.
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