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Measurement of PI4P Levels in Intact Chloroplasts Isolated from Arabidopsis thaliana
测量从拟南芥中分离的完整叶绿体中的PI4P含量   

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Abstract

Phosphatidylinositol 4-phosphate (PI4P), a major species of phosphoinositides, modulates many fundamental cellular processes. We have recently revealed that PI4P plays an important role in chloroplast division as a negative regulator. Despite its importance in chloroplasts, the content of PI4P in chloroplasts is very low and it is difficult to measure PI4P levels. In this protocol, we describe a simple method that we have developed for measurement of low level of PI4P in chloroplasts. Intact chloroplasts were isolated by a basic method using Percoll gradient centrifugation and acidic lipids were extracted from the isolated chloroplasts. The extracted acidic lipids including PI4P were spotted onto the membrane strip, which had been pre-spotted with PI4P standards and other phosphoinositides as negative controls. PI4P in the spot of acidic lipids on the membrane was detected using a PI4P binding protein.

Keywords: Phosphatidylinositol 4-phosphate(磷脂酰肌醇磷酸), PI4P(PI4P), Intact chloroplast(完整的叶绿体), Chloroplast isolation(叶绿体的分离)

Materials and Reagents

  1. Isolation of intact chloroplasts
    1. Miracloth (Merck Millipore Corporation, Calbiochem®, catalog number: 475855 )
    2. Gauze (Hakujuji Co., model: FC-gauze )
    3. Seedlings of Arabidopsis thaliana
      Wild-type (Columbia-0) plants, phosphatidylinositol 4-kinase (PI4K) α1 knockdown plants, pi4kβ2-1 mutants and PI4Kα1 knockdown plants of pi4kβ2-1 mutants were grown for 4 days on MS agar plates and then transferred onto agar plates with or without 5 μM dexamethazone (DEX) and grown for 1 week. The down-regulation of PI4Kα1 expression was induced by DEX treatments in PI4Kα1 knockdown plants and PI4Kα1 knockdown plants of pi4kβ2-1 mutants. For treatments with inhibitors, wild-type 4-d-old seedlings were transferred onto agar plates with PI4K inhibitors, 200 μM wortmannin (WM) or 25 μM phenylarsine oxide (PAO), or a phosphatidylinositol 3-kinase (PI3K) inhibitor, 50 μM LY294002 (LY), or without inhibitors (DMSO) and grown for 3 days.
    4. Percoll (GE Healthcare, Dharmacon, catalog number: 7-0891-01 )
    5. Bradford assay kit (Bio-Rad Laboratories, catalog number: 500-0006JA )
    6. 1x protease inhibitor cocktail (Nakarai, catalog number: 03969-21)
      Note: Currently, it is “Nacalai tesque, catalog number: 03969-21 ”.
    7. Sorbitol
    8. HEPES-KOH (pH 7.5)
    9. EDTA
    10. Grinding buffer (see Recipes)
    11. 80% Percoll or 40% Percoll (see Recipes)

  2. Extraction of PI4P
    1. 0.75 M Trichloroacetic acid (TCA) (Wako Pure Chemical Industries, Siyaku, catalog number: 203-04952 )
    2. 5% (w/v) TCA with 1 mM EDTA
    3. Methanol (Wako Pure Chemical Industries, Siyaku, catalog number: 137-01823 ): Chloroform (Wako Pure Chemical Industries, Siyaku, catalog number: 038-02601 ) (2:1, v/v)
    4. Methanol: chloroform: 12 N HCl (Wako Pure Chemical Industries, Siyaku, catalog number: 080-01066 ) (80:40:1, v/v/v)

  3. Measurement of PI4P levels
    1. PI(4)P Mass Strip Kit (Echelon Biosciences, catalog number: K-4000E )
    2. Albumin from bovine serum (BSA), fatty acid free (Wako Pure Chemical Industries, Siyaku, catalog number: 017-15146 )
    3. HRP substrate solution (Thermo Fisher Scientific, Pierce, catalog number: NCI32132 )
    4. Phosphate (pH 7.4)
    5. NaCl
    6. 0.1% (w/v) Tween-20
    7. Phosphate buffered saline (PBS) (see Recipes)
    8. PBST (see Recipes)

Equipment

  1. Homogenizer (Microtec Co., model: NS-51 )
  2. Centrifuge (TOMY SEIKO CO., models: MX-200 and GX-250 )
  3. Swing rotor (TS-7C)
  4. Paintbrush
  5. Pasteur pipette (Sansyo, Iwaki, catalog number: IK-PAS-9P )
  6. Vacuum dryer (Centrifuge evaporator) (Shimadzu Corporation, model: SPE-200 )
  7. Sonicator (SHARP CORPORATION, model: UT-106 )

Procedure

  1. Isolation of intact chloroplasts
    1. Sampling 0.5-1 g of seedlings of Arabidopsis thaliana. Whole seedlings for one-week-old plants or shoots for eleven-day-old plants were used.
    2. Add 6 ml of grinding buffer and homogenize with homogenizer 3-5 times for 3 sec on ice.
    3. Filtrate the homogenate through 2 layers of gauze and a layer of Miracloth (put gauzes on the Miracloth).
    4. Centrifuge at 2,500 x g for 15 min at 4 °C. Discard the supernatant and gently resuspend the pellet in 1.5 ml of grinding buffer with a paintbrush. Brush the pellet gently with paintbrush little by little to avoid breaking of intact chloroplasts. Note that pipetting the pellet breaks chloroplasts.
    5. Add 1.5 ml of grinding buffer and 1 ml of 80% Percoll and gently mix. (Final concentration of Percoll is 20%.)
    6. Prepare Percoll step gradients. Layering first 3 ml of 80% Percoll and then 5 ml of 40% Percoll in 15 ml centrifuge tubes. Put the tip of pipette on the surface and slowly lay 40% Percoll to avoid disturbance of the gradient. See Figure 1.
    7. Lay 4 ml of crude chloroplast suspension (the results of step A5) on the top of Percoll gradients.
    8. Centrifuge at 3,200 x g for 30 min at 4 °C using a swing rotor. Set speed of acceleration and deceleration at minimum.
    9. Collect a green band with a Pasteur pipette corresponding to intact chloroplasts at the interface between 40% and 80% to 2 ml tubes. Remove carefully the upper band (broken chloroplasts) and transfer lower band to 2 ml tubes with a Pasteur pipette. See Figure 1.
    10. Add grinding buffer up to 2 ml and centrifuge at 700 x g for 5 min at 4 °C.
    11. Discard the supernatant and wash the pellet two times by resuspension in 1 ml of grinding solution and centrifugation at 700 x g for 5 min at 4 °C.
    12. Suspend the pellet (intact chloroplasts) in 0.3 ml of grinding buffer.
    13. Take a part of the samples for measurement of protein concentration (step B9).


      Figure 1. Isolation of intact chloroplasts with Percoll gradient centrifugation. 1. Lay 40% Percoll on 80% Percoll; 2. Lay crude chloroplast suspension on 40% Percoll; 3. The tube before centrifugation; 4. The tube after centrifugation; 5. Remove the upper band (broken chloroplasts); 6. Collect the lower band (intact chloroplasts).

  2. Extraction of PI4P
    1. Add 750 μl of ice cold 0.75 M TCA to 250 μl of intact chloroplast samples. Vortex and incubate on ice for 5 min.
    2. Centrifuge at 700 x g for 5 min at 4 °C and discard the supernatant. Add 1 ml of 5% TCA with 1 mM EDTA to the pellet and vortex for 30 sec.
    3. Centrifuge at 700 x g for 5 min at room temperature and discard the supernatant. Add 1 ml of methanol:chloroform (2:1) to the pellet for extraction of neutral lipids and vortex 3 times for 30 sec at an interval of 5 min at room temperature.
    4. Centrifuge at 700 x g for 5 min at room temperature and discard the supernatant. Add 1 ml of methanol:chloroform (2:1) to the pellet for extraction of neutral lipids again and vortex for 1 min at room temperature.
    5. Centrifuge at 700 x g for 5 min at room temperature and discard the supernatant. Add 750 μl of methanol:chloroform:12 N HCl (80:40:1) to the pellet for extraction of acidic lipids and vortex 4 times for 30 sec at an interval of 5 min.
    6. Add 250 μl of chloroform and 450 μl of 0.1 M HCl and vortex.
    7. Centrifuge at 700 x g for 5 min, collect the lower phase and transfer it to new tubes.
    8. Dry samples in a vacuum dryer.
    9. Measure protein concentration. Add 1-10 μl of samples (step A13) into 1 ml of Bradford assay reagents (1:5 dilution). Incubate for 5 min at room temperature and measure absorbance at 595 nm. Measure absorbance of standard protein samples too. Make a standard curve using standard protein samples and calculate the PI4P concentration according to the reads. The concentration of protein is usually 0.3-0.8 mg/ml.

  3. Measurement of PI4P levels
    1. Add 15 μl of chloroform: methanol: water (1:2:0.8, v/v/v) to the sample. Vortex for 30 sec and sonicate (100% output) in icy water bath for 5 min. Vortex again for 30 sec.
    2. Centrifuge 15,000 x g for 5 min and transfer 10 μl of supernatant to new tube. Do not suck the pellet at the bottom of tube.
    3. Spot samples onto a PI(4)P Strip. Spot 1 μl of sample at a time. Before spotting sample again in the same area, dry the spot. After all samples are spotted, dry the strip for 30 min at room temperature. See Figure 2.
    4. Incubate the membrane with blocking solution (3% fatty acid-free BSA in PBS) for 1 h at room temperature with gentle agitation.
    5. Incubate the membrane with 5 ml of blocking solution containing 1 vial of PI(4)P Detector (2.5 μg protein) for 1 h at room temperature with gentle agitation.
    6. Discard the solution and wash the membrane with 10 ml of PBST three times at room temperature with gentle agitation for 5 min each.
    7. Incubate the washed membrane with 5 ml of blocking solution containing 75 μl of secondary detector for 1 h at room temperature with gentle agitation.
    8. Discard the solution and wash the membrane three times with 10 ml of PBST at room temperature with gentle agitation for 5 min each.
    9. Incubate the washed membrane with enough ECL solution to cover the membrane at room temperature for 5 min.
    10. Wash the membrane with water three times to stop the reaction and dry the membrane.
    11. Scan the membrane to record the obtained data. See Figure 2.

Representative data


Figure 2. Representative results of PI4P detection assay. Samples extracted from intact chloroplasts were spotted left side of the PI(4)P Strip. PI4P standards (20, 15, 10, 5, 4, 2, 1, and 0.5 pmol of PI4P) and phosphoinositide controls were pre-spotted on the membrane. Lipids extracted from isolated chloroplasts of wild-type plants, phosphatidylinositol 4-kinase (PI4K) α1 knockdown plants, pi4kβ2-1 mutants and PI4Kα1 knockdown plants of pi4kβ2-1 mutants treated with/without 5 μM dexamethasone (DEX) were spotted onto the membrane. The down-regulation of PI4Kα1 expression was induced by DEX treatments in PI4Kα1 knockdown plants and PI4Kα1 knockdown plants of pi4kβ2-1 mutants. Lipids extracted from isolated chloroplasts of wild-type plants treated with/without PI4K inhibitors, 200 μM wortmannin (WM) or 25 μM phenylarsine oxide (PAO), or a phosphatidylinositol 3-kinase (PI3K) inhibitor, 50 μM LY294002 (LY), were also spotted. Sample spots are: 1. Wild-type plants without DEX; 2. Wild-type plants treated with DEX; 3. PI4Kα1 knockdown plants without DEX; 4. PI4Kα1 knockdown plants treated with DEX; 5. pi4kβ2-1 mutants without DEX; 6. pi4kβ2-1 mutants treated with DEX; 7. PI4Kα1 knockdown plants of pi4kβ2-1 mutants treated without DEX; 8. PI4Kα1 knockdown plants of pi4kβ2-1 mutants treated with DEX; 9. Wild-type plants without inhibitor; 10. Wild-type plants with WM; 11. Wild-type plants with PAO; 12. Wild-type plants with LY. The abbreviations used are: PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI5P, phosphatidylinositol 5-phosphate; PI(3, 4)P2, phosphatidylinositol 3, 4-bisphosphate; PI(3, 5)P2, phosphatidylinositol 3, 5-bisphosphate; PI(4, 5)P2, phosphatidylinositol 4, 5-bisphosphate; PI(3, 4, 5)P3, phosphatidylinositol 3, 4, 5-triphosphate.

Notes

  1. The samples and Percoll gradient tubes should be kept on ice during procedure A.
  2. Do not homogenize samples too much. Three to five times for 3 sec are usually enough. Longer homogenization results in lower yield of intact chloroplasts.
  3. After step B3, do not keep samples on ice.
  4. Intactness of chloroplasts can be assessed by phase contrast microscope. Intact chloroplasts are surrounded by bright halos of light. Broken chloroplasts look dark. See Figure 3 of Klinkenberg (2014).
  5. Extraction of neutral lipids should be done twice for removing chlorophyll.
  6. Do not suck the pellet at step C2. It contains left over proteins from extraction steps and makes hydrophobic film on the membrane. The hydrophobic film prevents binding of PI4P detector to PI4P. See Figure 3.


    Figure 3. An example of a failed PI4P detection assay. Lipids were spotted onto the membrane together with the debris left over in the extraction step (the pellet at step C2). It prevented binding of PI(4)P Detector to PI4P and thus spots were detected as rings instead of circles.

Recipes

  1. Grinding buffer
    0.33 M sorbitol
    30 mM HEPES-KOH (pH 7.5)
    2 mM EDTA
    1x protease inhibitor cocktail
    Autoclave without protease inhibitor cocktail
    Protease inhibitor cocktail is added before use.
  2. 80% or 40% Percoll
    30 mM HEPES-KOH (pH 7.5)
    0.33 M sorbitol
    2 mM EDTA
    Percoll (80% or 40%, v/v)
  3. PBS
    10 mM phosphate (pH 7.4)
    150 mM NaCl
    Sterilized by autoclave
  4. PBST
    PBS
    0.1% (w/v) Tween-20

Acknowledgments

This protocol was adapted from the original work (Okazaki et al., 2015) to provide the detailed procedures. This work was supported by JSPS KAKENHI Grant Number 26840089.

References

  1. Klinkenberg, J. (2014). Extraction of chloroplast proteins from transiently transformed Nicotiana benthamiana leaves. Bio-protocol 4(18): e1238.
  2. Nakanishi, H., Suzuki, K., Kabeya, Y. and Miyagishima, S. Y. (2009). Plant-specific protein MCD1 determines the site of chloroplast division in concert with bacteria-derived MinD. Curr Biol 19(2): 151-156.
  3. Okazaki, K., Miyagishima, S. Y. and Wada, H. (2015). Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in Arabidopsis. Plant Cell 27(3): 663-674.

简介

磷脂酰肌醇4-磷酸(PI4P)是磷酸肌醇的主要种类,调节许多基本的细胞过程。 我们最近揭示PI4P在叶绿体分裂作为负调节器中发挥重要作用。 尽管其在叶绿体中的重要性,PI4P在叶绿体中的含量非常低,并且难以测量PI4P水平。 在这个协议,我们描述了一个简单的方法,我们已经开发测量叶绿体中的低水平的PI4P。 通过使用Percoll梯度离心的碱性方法分离完整的叶绿体,并从分离的叶绿体中提取酸性脂质。 将包括PI4P的提取的酸性脂质点样在膜条上,其已经用PI4P标准品和其它磷酸肌醇作为阴性对照预先点样。 使用PI4P结合蛋白检测膜上酸性脂质斑点中的PI4P。

关键字:磷脂酰肌醇磷酸, PI4P, 完整的叶绿体, 叶绿体的分离

材料和试剂

  1. 完整叶绿体的分离
    1. Miracloth(Merck Millipore Corporation,Calbiochem ,目录号:475855)
    2. Gauze(Hakujuji Co.,型号:FC-gauze)
    3. 拟南芥的幼苗
      野生型(Columbia-0)植物,磷脂酰肌醇4-激酶(

      PI4K )α1敲低植物,pi4kβ2-1< em4突变体和< im>PI4Kα1</p>敲低植物在MS琼脂平板上生长4天,然后 转移到具有或不具有5μM地塞米松(DEX)的琼脂板上, 并生长1周。 PI4Kα1表达的下调是 通过在PI4Kα1敲除植物中的DEX处理和在p14Kβ2-1突变体中产生的PI4Kα1敲除植物诱导。对于用抑制剂的治疗, 将野生型4日龄幼苗转移到具有PI4K的琼脂平板上 抑制剂,200μM渥曼青霉素(WM)或25μM氧化苯基砷(PAO) ?磷脂酰肌醇3-激酶(PI3K)抑制剂,50μMLY294002(LY), 或不含抑制剂(DMSO)并生长3天。
    4. Percoll(GE Healthcare,Dharmacon,目录号:7-0891-01)
    5. Bradford测定试剂盒(Bio-Rad Laboratories,目录号:500-0006JA)
    6. 1x蛋白酶抑制剂混合物(Nakarai,目录号:03969-21)
      注意:目前,它是"Nacalai tesque,目录号:03969-21"。
    7. 山梨醇
    8. HEPES-KOH(pH7.5)
    9. EDTA
    10. 研磨缓冲液(参见配方)
    11. 80%Percoll或40%Percoll(参见配方)

  2. 提取PI4P
    1. 0.75M三氯乙酸(TCA)(Wako Pure Chemical Industries,Siyaku,目录号:203-04952)
    2. 5%(w/v)TCA和1mM EDTA
    3. 甲醇(Wako Pure Chemical Industries,Siyaku,目录号: 137-01823):氯仿(Wako Pure Chemical Industries,Siyaku,目录 编号:038-02601)(2:1,v/v)
    4. 甲醇:氯仿:12N HCl(Wako Pure Chemical Industries,Siyaku,目录号:080-01066)(80:40:1,v/v/v)

  3. PI4P水平的测量
    1. PI(4)P Mass Strip Kit(Echelon Biosciences,目录号:K-4000E)
    2. 来自牛血清(BSA),不含脂肪酸的白蛋白(Wako Pure Chemical Industries,Siyaku,目录号:017-15146)
    3. HRP底物溶液(Thermo Fisher Scientific,Pierce,目录号:NCI32132)
    4. 磷酸盐(pH 7.4)
    5. NaCl
    6. 0.1%(w/v)Tween-20
    7. 磷酸盐缓冲盐水(PBS)(见Recipes)
    8. PBST(参见配方)

设备

  1. 均质机(Microtec Co.,型号:NS-51)
  2. 离心机(TOMY SEIKO CO。,型号:MX-200和GX-250)
  3. 摆动转子(TS-7C)
  4. 油漆刷
  5. 巴斯德吸管(Sansyo,Iwaki,目录号:IK-PAS-9P)
  6. 真空干燥器(离心蒸发器)(Shimadzu Corporation,型号:SPE-200)
  7. 超声波仪(SHARP CORPORATION,型号:UT-106)

程序

  1. 完整叶绿体的分离
    1. 取0.5-1g拟南芥的幼苗。整个幼苗 使用1周龄植物或11天龄植物的芽。
    2. 加入6ml研磨缓冲液并用匀浆器在冰上匀化3-5次3秒。
    3. 通过2层纱布和一层Miracloth(放在Miracloth上的纱布)过滤匀浆。
    4. 在4℃下以2500xg离心15分钟。弃去上清液 并轻轻地将沉淀重悬在1.5ml的研磨缓冲液中 画笔。用刷子轻轻地刷小丸一点点 避免破坏完整的叶绿体。注意移液沉淀 打破叶绿体。
    5. 加入1.5毫升研磨缓冲液和1毫升80%Percoll,轻轻混合。 (Percoll的最终浓度为20%。)
    6. 准备Percoll步梯度。首先分层3ml的80%Percoll 然后在15ml离心管中加入5ml 40%Percoll。把尖头 移液管表面,并缓慢放置40%Percoll,以避免干扰 的梯度。见图1.
    7. 在Percoll梯度顶部放置4ml粗制叶绿体悬浮液(步骤A5的结果)
    8. 使用摆动转子在4℃下以3,200×g离心30分钟。将加速和减速的速度设置为最小。
    9. 用对应于完整的巴斯德吸管收集绿色带 叶绿体在40%和80%至2ml管之间的界面。去掉 仔细地上带(破碎的叶绿体)和转移下带 到具有巴斯德移液管的2ml管。见图1.
    10. 加入研磨缓冲液至多2ml,并在4℃下以700×g离心5分钟。
    11. 弃去上清液,洗涤沉淀两次 再悬浮于1ml研磨溶液中并在4℃下以700×g离心5分钟。
    12. 将颗粒(完整叶绿体)悬浮在0.3ml研磨缓冲液中。
    13. 取一部分样品测量蛋白质浓度(步骤B9)

      图1.用Percoll梯度分离完整叶绿体 离心。 1.将40%Percoll置于80%Percoll上;放置粗 叶绿体悬浮液在40%Percoll上; 3.管前 离心; 4.离心后管; 5.取下上部 带(断裂的叶绿体); 6.收集下部带(完好无损 叶绿体)。

  2. 提取PI4P
    1. 加入750微升冰冷的0.75 M TCA到250微升完整的叶绿体样品。涡旋并在冰上孵育5分钟。
    2. 在4℃下以700×g离心5分钟,弃去上清液。 ?加入1ml含有1mM EDTA的5%TCA至沉淀并涡旋30秒。
    3. 在室温下以700×g离心5分钟,弃去 上清液。向沉淀中加入1ml甲醇:氯仿(2:1) 提取中性脂质并涡旋3次,每次30秒 间隔5分钟,室温。
    4. 700离心机离心。 ?5分钟,弃去上清液。加入1毫升 甲醇:氯仿(2:1)洗脱,以提取中性脂质 ?并在室温下涡旋1分钟。
    5. 离心机 700×g在室温下处理5分钟,弃去上清液。加 向沉淀中加入750μl甲醇:氯仿:12N HCl(80:40:1) 提取酸性脂质并涡旋4次,每次30秒 ?的5分钟。
    6. 加入250μl氯仿和450μl0.1M HCl并涡旋。
    7. 以700×g离心5分钟,收集下层相并将其转移到新试管中。
    8. 在真空干燥器中干燥样品。
    9. 测量蛋白浓度。加入1-10μl样品(步骤A13) 到1ml的Bradford测定试剂(1:5稀释)中。孵育5分钟 并测量595nm处的吸光度。测量吸光度 ?的标准蛋白样品。使用标准品制作标准曲线 蛋白质样品并计算PI4P浓度 读取。蛋白质的浓度通常为0.3-0.8mg/ml。

  3. PI4P水平的测量
    1. 向样品中加入15μl氯仿:甲醇:水(1:2:0.8,v/v/v)。 ?涡旋30秒,并在冰水浴中声处理(100%输出)5 min。再次涡旋30秒。
    2. 离心15,000×g 5分钟 并将10μl上清液转移到新试管中。不要吸取沉淀 ?管的底部。
    3. 将样品点样到PI(4)P条上。点1μl ?的样品。在同一区域再次点样前,干燥 ?位置。在所有样品点样后,在30℃下干燥该条30分钟 室内温度。见图2.
    4. 孵育膜 封闭溶液(PBS中3%无脂肪酸的BSA)在室温下封闭1小时 温和搅拌。
    5. 孵育膜与5 ml含有1小瓶PI(4)P检测剂(2.5μg 蛋白)在室温下温和搅拌1小时。
    6. 弃去溶液并用10ml PBST洗涤膜三次 次,在室温下温和搅拌5分钟。
    7. 用5ml含有的封闭溶液孵育洗涤过的膜 75μl次级检测器在室温下温和1小时 搅动。
    8. 弃去溶液并洗涤膜三 用10ml PBST在室温下温和搅拌5次 ?min。
    9. 用足够的ECL溶液孵育洗涤的膜,以在室温下覆盖膜5分钟
    10. 用水洗涤膜三次以停止反应并干燥膜
    11. 扫描膜以记录所获得的数据。参见图2。

代表数据


图2. PI4P检测测定的代表性结果从完整叶绿体提取的样品点在PI(4)P条带的左侧。将PI4P标准品(20,15,10,5,4,2,1和0.5pmol的PI4P)和磷酸肌醇对照物预点在膜上。从野生型植物的分离的叶绿体中提取的脂质,磷脂酰肌醇4-激酶(

PI4K )α1敲低植物,pi4kβ2-1<用/不用5μM地塞米松(DEX)处理的pI4kβ2-1突变体和< em>突变体和< em>PI4Kα1敲低植物点在膜上。通过在PI4Kα1敲低植物中的DEX处理和在p14Kβ1-1敲除植物中的PI4Kα1敲低植物诱导PI4Kα1表达的下调, em>突变体。从用/不用PI4K抑制剂,200μM渥曼青霉素(WM)或25μM氧化苯氧胺(PAO)或磷脂酰肌醇3-激酶(PI3K)抑制剂,50μMLY294002(LY)处理的野生型植物的分离的叶绿体中提取的脂质,也被发现了。样品点是:1.没有DEX的野生型植物; 2.用DEX处理的野生型植物; 3.没有DEX的PI4Kα1敲低植物; 4.用DEX处理的PI4Kα1敲低植物; 5.没有DEX的pi4kβ2-1突变体; 6.用DEX处理的p14kβ2-1突变体; 7.不使用DEX处理的PI4Kα1敲除植物的p14kβ2-1突变体; 8.用DEX处理的pI4kβ2-1突变体的PI4Kα1敲低植物; 9.无抑制剂的野生型植物; 10.具有WM的野生型植物; 11.具有PAO的野生型植物; 12.具有LY的野生型植物。使用的缩写是:PI,磷脂酰肌醇; PI3P,磷脂酰肌醇3-磷酸; PI5P,磷脂酰肌醇5-磷酸; PI(3,4)P 2,磷脂酰肌醇3,4-二磷酸酯; PI(3,5)P 2,磷脂酰肌醇3,5-二磷酸酯; PI(4,5)P 2,磷脂酰肌醇4,5-二磷酸酯; PI(3,4,5)P 3,磷脂酰肌醇3,4,5-三磷酸。

笔记

  1. 在操作A期间,样品和Percoll梯度管应保持在冰上
  2. 不要使样品过于均匀。三到五次,持续3秒通常就足够了。较长的匀浆导致完整叶绿体的产量较低。
  3. 在步骤B3之后,不要将样品保持在冰上。
  4. 叶绿体的完整性可以通过相差显微镜评估。完整的叶绿体被明亮的光晕包围。破碎的叶绿体看起来很黑。参见Klinkenberg图(2014)。
  5. 提取中性脂质应进行两次以去除叶绿素。
  6. 在步骤C2不要吸取沉淀。它含有来自提取步骤的剩余蛋白质,并在膜上形成疏水膜。疏水膜防止PI4P检测器与PI4P的结合。见图3.


    图3.失效的PI4P检测测定的实例将脂质与在提取步骤中剩余的碎片(步骤C2中的沉淀)一起点在膜上。它防止PI(4)P检测器与PI4P结合,因此将斑点检测为环而不是圆。

食谱

  1. 研磨缓冲器
    0.33M山梨醇 30mM HEPES-KOH(pH7.5) 2mM EDTA 1x蛋白酶抑制剂混合物
    无蛋白酶抑制剂混合物的高压灭菌器
    在使用前加入蛋白酶抑制剂混合物
  2. 80%或40%Percoll
    30mM HEPES-KOH(pH7.5) 0.33M山梨醇 2mM EDTA Percoll(80%或40%,v/v)
  3. PBS
    10mM磷酸盐(pH7.4) 150mM NaCl 高压灭菌器灭菌
  4. PBST
    PBS
    0.1%(w/v)Tween-20

致谢

该协议改编自原始工作(Okazaki等人,2015),以提供详细的程序。这项工作得到JSPS KAKENHI Grant号26840089的支持。

参考文献

  1. Klinkenberg,J。(2014)。 从瞬时转化的烟草叶中提取叶绿体蛋白。 生物协议 4(18):e1238
  2. Nakanishi,H.,Suzuki,K.,Kabeya,Y.and Miyagishima,S.Y。(2009)。 植物特异性蛋白MCD1决定了与细菌衍生的MinD一起的叶绿体分裂位点。 a> Curr Biol 19(2):151-156
  3. Okazaki,K.,Miyagishima,S.Y.and Wada,H。(2015)。 磷脂酰肌醇4-磷酸负调节拟南芥中的叶绿体分裂。 植物细胞 27(3):663-674。
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引用:Okazaki, K., Miyagishima, S. and Wada, H. (2016). Measurement of PI4P Levels in Intact Chloroplasts Isolated from Arabidopsis thaliana. Bio-protocol 6(3): e1719. DOI: 10.21769/BioProtoc.1719.
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