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Thylakoids are a formation of flattened membrane vesicles and protein complexes found in cyanobacteria, algae and plants. In the chloroplasts of land plants the thylakoid membrane systems form a network of densely packed stacks called grana lamellae, which are connected by unstacked stroma lamellae. Photosystem II is mainly localized in the appressed grana region, while photosystem I and the ATP synthase complexes are enriched in the stroma lamellae. The cytochrome b6/f complex is distributed laterally throughout both stacked and unstacked membrane regions. The photosynthetic complexes consist of integral and peripheral proteins. The first part of this protocol (A) shows how to fractionate thylakoids into grana and stroma lamellae. The second part of this protocol (B) shows how to distinguish between strong hydrophobic integral membrane associations and weak electrostatic membrane and/or membrane complex associations. As it is necessary to specifically detect the protein of interest in the fractions, a specific antibody raised against the protein of interest or a complemented null mutant of a structural component expressing a tagged fusion protein would be of great advantage. The last part of this protocol (C) shows, how to investigate the topology of integral and peripheral proteins. This method requires a specific antibody for the protein of interest. For integral membrane proteins peptide-specific antibodies or epitope-tagged versions are required. The protocol is suitable for the investigation of low molecular weight proteins (LMW) below 5 kDa (Torabi et al., 2014).

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Localization and Topology of Thylakoid Membrane Proteins in Land Plants
陆生植物类囊体膜蛋白的定位和拓扑结构

植物科学 > 植物生物化学 > 蛋白质 > 结构
作者: Salar Torabi
Salar TorabiAffiliation: Biozentrum der LMU München, Department Biologie I, Planegg-Martinsried, Germany
For correspondence: salar.torabi@biologie.uni-muenchen.de
Bio-protocol author page: a1885
Magdalena Plöchinger
Magdalena PlöchingerAffiliation: Biozentrum der LMU München, Department Biologie I, Planegg-Martinsried, Germany
Bio-protocol author page: a1886
 and Jörg Meurer
Jörg MeurerAffiliation: Biozentrum der LMU München, Department Biologie I, Planegg-Martinsried, Germany
Bio-protocol author page: a1887
Vol 4, Iss 24, 12/20/2014, 3619 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1363

[Abstract] Thylakoids are a formation of flattened membrane vesicles and protein complexes found in cyanobacteria, algae and plants. In the chloroplasts of land plants the thylakoid membrane systems form a network of densely packed stacks called grana lamellae, which are connected by unstacked stroma lamellae. Photosystem II is mainly localized in the appressed grana region, while photosystem I and the ATP synthase complexes are enriched in the stroma lamellae. The cytochrome b6/f complex is distributed laterally throughout both stacked and unstacked membrane regions. The photosynthetic complexes consist of integral and peripheral proteins. The first part of this protocol (A) shows how to fractionate thylakoids into grana and stroma lamellae. The second part of this protocol (B) shows how to distinguish between strong hydrophobic integral membrane associations and weak electrostatic membrane and/or membrane complex associations. As it is necessary to specifically detect the protein of interest in the fractions, a specific antibody raised against the protein of interest or a complemented null mutant of a structural component expressing a tagged fusion protein would be of great advantage. The last part of this protocol (C) shows, how to investigate the topology of integral and peripheral proteins. This method requires a specific antibody for the protein of interest. For integral membrane proteins peptide-specific antibodies or epitope-tagged versions are required. The protocol is suitable for the investigation of low molecular weight proteins (LMW) below 5 kDa (Torabi et al., 2014).
Keywords: Stroma(基质), Grana(基粒), Photosystem(光系统), Topology(拓扑), Thylakoid(类囊体)

[Abstract] 类囊体是在蓝细菌,藻类和植物中发现的扁平膜囊泡和蛋白质复合物的形成。在陆地植物的叶绿体中,类囊体膜系统形成称为颗粒薄片的密集堆叠的网络,其通过未堆叠的基质薄片连接。光系统II主要定位在贴壁的颗粒区域,而光系统I和ATP合酶复合物富集基质层。细胞色素e / f 复合物横向分布在堆叠和未堆叠的膜区域。光合复合物由整合蛋白和外周蛋白组成。本协议(A)的第一部分显示如何分裂类囊体成grana和基质层。该方案的第二部分(B)显示了如何区分强疏水整合膜缔合和弱静电膜和/或膜复合物缔合。由于必须特异性检测级分中的目标蛋白,针对目的蛋白产生的特异性抗体或表达标记融合蛋白的结构组分的互补无效突变体将是非常有利的。本协议(C)的最后一部分显示,如何调查内在和外围蛋白的拓扑。该方法需要针对目标蛋白的特异性抗体。对于内在膜蛋白,需要肽特异性抗体或表位标记的形式。该方案适合于低于5kDa的低分子量蛋白质(LMW)的研究(Torabi等人,2014)。

Material and Reagents

  1. Freshly isolated thylakoids
  2. Digitonin (water soluble) (SERVA Electrophoresis GmbH, catalog number: 19551.02 )
  3. Thermolysin from Bacillus thermoproteolyticus (Calbiochem®, catalog number: 58656 )
  4. NaBr (Sigma-Aldrich, catalog number: S-9756 )
  5. NaSCN (Merck KGaA, catalog number: 6627 )
  6. Na2CO3 (Merck KGaA, catalog number: 6392 )
  7. NaOH (Roth North America, catalog number: 9097.2 )
  8. Sucrose (Roth North America, catalog number: 9097.2 )
  9. EDTA (SERVA Electrophoresis GmbH, catalog number: 11280 )
  10. HEPES (Roth North America, catalog number: 9105.3 )
  11. Na2HPO4 (Roth North America, catalog number: 4984.3 )
  12. NaH2PO4 (Roth North America, catalog number: K300.2 )
  13. 0.1 M sodium phosphate buffer (see Recipes)
  14. Fractionation buffer (see Recipes)
  15. HS buffer (see Recipes)
  16. 0.4% digitonin solution (see Recipes)
  17. Thermolysin stock solution (40 mg/ml) in HS buffer (see Recipes)
  18. Salt containing HS buffers (see Recipes)

Equipment

  1. Centrifuges (Beckmann Coulter, model: Avanti J-25 ; Eppendorf, model: 5430 R )
  2. Ultracentrifuge (Beckmann Coulter, model: Optima LE-80K )
  3. Sonifier (Branson, model: B-12 )
  4. Photometer (Amersham biosciences, model: UltraspeTM 3100 pro )

Procedure

  1. Fractionation of thylakoid membranes
    1. To solubilize the thylakoids at the grana margins mix 5 ml of freshly isolated thylakoids (0.8 mg chlorophyll/ml) in fractionation buffer with 5 ml of the 0.4% digitonin solution and incubate 2 min at RT. Prevent sedimentation of the thylakoid solution by slightly agitating.
    2. Stop the solubilization with the addition of 90 ml ice cold fractionation buffer.
    3. Centrifuge the solution for 15 min (10,000 x g, 4 °C). Carefully keep the supernatant for the next step and try to avoid contaminations from the pellet. Dissolve the pellet (grana fraction) in 2 ml fractionation buffer and store on ice.
    4. Centrifuge the supernatant of step A3 for 30 min (40,000 x g, 4 °C). Carefully remove the upper part of the supernatant for step A5 and leave about 3 cm of the solution to avoid contamination of the stroma lamellae fraction. (Optional) dissolve the pellet in the remaining supernatant and keep the intermittent fraction on ice.
      Pellet stroma lamellae by ultracentrifugation of the supernatant of step A4 for 60 min (100,000 x g, 4 °C). Carefully remove the supernatant and dissolve the pellet in fractionation buffer. Try to keep the concentration as high as possible to avoid additional ultracentrifugation steps when adjusting the fractions to the desired chlorophyll concentrations. For storage of native thylakoid fractions (-20 °C or -80 °C) high chlorophyll concentrations around 2-4 mg/ml are recommended. For SDS-PAGE analysis final concentrations from 0.25 to 1 µg Chl/µl are used depending on the protein of interest. As a general rule use a lower final protein concentration for bigger membrane proteins with many cysteins to reduce (e.g. CP47, PsaA). Higher protein concentrations are suitable for low molecular weight proteins to reduce the sample volume and to avoid an expanded loading buffer front which could interfere with the migration of very small proteins.
    5. Measure the chlorophyll contents of the fractions in 80% acetone and adjust to equal amounts of µg chlorophyll/µl.
    6. Analyze the fractionation by SDS-PAGE (Figure 1).

  2. Salt treatment of thylakoid membranes
    1. Dissolve freshly isolated thylakoid membranes in HS buffer (0.5 mg chlorophyll/ml) and in the salt containing HS buffers.
    2. Incubate the samples for 30 min on ice.
    3. Dilute the samples with two volumes of HS buffer.
    4. Separate into pellet and soluble fraction by 10 min centrifugation (20,000 x g, 4°C).
    5. Analyze the fractions by SDS-PAGE.

  3. Thermolysin treatment of thylakoid membranes
    1. Dissolve freshly isolated thylakoid membranes in HS buffer (0.5 mg chlorophyll/ml).
    2. To produce 50% inside-out and 50% right-side out vesicles apply ultrasonic pulses (10-30 sec, 10-30 times) to the solution on ice. Wait about 20 sec in between the pulses for cooling.
    3. Add the protease thermolysin to a final concentration of 100 µg/ml to the untreated thylakoid membrane solution and the inside- and right-side out vesicles.
    4. Take probes at different time points (for example 0, 1, 2, 5 min) and immediately stop the digestion by adding EDTA to a final concentration of 20 mM.
    5. Wash probes in HS buffer containing 20 mM EDTA.
    6. Compare the digestion of thylakoids and inside- and right-side out vesicles by SDS-PAGE.

Representative data



Figure 1. Thylakoid system


Figure 2. Scheme of the fractionation procedure (A)


Figure 3. Coomassie staining of the thylakoid fractionation (A). Thylakoid membranes (T) were fractionated into stroma lamellae (S), intermittent fraction (I), and grana lamellae (G), and the separated proteins were subsequently stained with Coomassie to judge the purity of the fractions. PSI proteins PsaA and PsaB as well as the ATP synthase subunits CFo α/β were highly enriched in the stroma lamellae and only small amounts were present in the grana membranes. The PSII proteins CP47, CP43, D1, D2 and the antenna proteins of the light harvesting complex of PSII (LHCII) were predominantly present in grana fractions but almost lacking in the stroma lamellae fraction. The intermittent fraction contained proteins of both photosystems. An equal amount of chlorophyll (5 µg) was loaded.


Figure 4. Washing experiment (B). Thylakoid membranes treated with different salt-containing buffers were fractionated into pellet (P) and supernatant (S) and separated by SDS-PAGE. The separated proteins were analyzed by Coomassie staining and immunoblot analysis. The dissociation of the hydrophilic ATP synthase subunits CFo α/β from the membrane can be identified by Coomassie staining under all salt conditions used. In contrast the hydrophobic light harvesting complex of PSII (LHCII) and the PSI core subunits PsaA/B could not be released from the pellet fraction. Specific antisera were used to identify the peripheral lumenal PsbO protein, which is associated with PSII. The PsbO protein could be released completely from the membrane only under stringent salt conditions (2 M NaSCN and 0.1 M NaOH).


Figure 5. Scheme of the protease treatment (C)


Figure 6. Topology studies (C). Untreated and sonicated thylakoids were incubated with thermolysin and subjected to immunodecoration using PsbO and PsaE antisera. In the untreated thylakoids the lumenal PsbO is protected from thermolysin treatment, while the stromal exposed PsaE is degraded. In the sonicated thylakoids, which form about 50% inside-out vesicles, PsbO is partially degraded, while PsaE is partially protected.

Notes

  1. To dissolve a large thylakoid pellet as fast and gentle as possible use a fine paint brush.
  2. For the separation of thylakoid membrane proteins by SDS-PAGE and especially proteins ≤ 10 kDa Tricine-SDS-PAGE is highly recommended for reference (Schägger, 2006).
  3. For immuno-blot analysis of low molecular weight thylakoid membrane proteins use 0.2 µM PVDF or 0.1 µM nitrocellulose membrane and shorten the transfer time.

Recipes

  1. 0.1 M sodium phosphate buffer (pH 7.4)
    1 M HEPES-NaOH (pH 8.0)
    0.5 M EDTA (pH 8)
  2. Fractionation buffer
    100 mM sucrose
    10 mM sodium-phosphate buffer (pH 7.4)
    5 mM MgCl2
    5 mM NaCl
  3. HS buffer
    0.1 M sucrose
    10 mM HEPES-NaOH (pH 8.0)
  4. 0.4% digitonin solution
    Dissolve 40 mg digitonin in 10 ml H2O by mixing and heating
    Solution can be stored at -20 °C
  5. Thermolysin stock solution (40 mg/ml) in HS buffer
    Stock can be stored at -20 °C
  6. Salt containing HS buffers
    2 M NaBr in HS buffer
    2 M NaSCN in HS buffer
    0.1 M Na2CO3 in HS buffer
    0.1 mM NaOH in HS buffer A

Acknowledgments

This protocol was adapted or modified from previous work by Karnauchov et al. (1997). This research was supported by the German Science Foundation (Deutsche Forschungsgemeinschaft (ME 1794) to J. M.

References

  1. Armbruster, U., Zuhlke, J., Rengstl, B., Kreller, R., Makarenko, E., Rühle, T., Schünemann, D., Jahns, P., Weisshaar, B., Nickelsen, J. and Leister, D. (2010). The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II assembly. Plant Cell 22(10): 3439-3460.
  2. DalCorso, G., Pesaresi, P., Masiero, S., Aseeva, E., Schünemann, D., Finazzi, G., Joliot, P., Barbato, R. and Leister, D. (2008). A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis. Cell 132(2): 273-285.
  3. Karnauchov, I., Herrmann, R. G. and Klosgen, R. B. (1997). Transmembrane topology of the Rieske Fe/S protein of the cytochrome b6/f complex from spinach chloroplasts. FEBS Lett 408(2): 206-210.
  4. Torabi, S., Umate, P., Manavski, N., Plöchinger, M., Kleinknecht, L., Bogireddi, H., Herrmann, R. G., Wanner, G., Schröder, W. P. and Meurer, J. (2014). PsbN is required for assembly of the photosystem II reaction center in Nicotiana tabacum. Plant Cell 26(3): 1183-1199.
  5. Schagger, H. (2006). Tricine-SDS-PAGE. Nat Protoc 1(1): 16-22.

材料和试剂

  1. 新鲜隔离的类囊体
  2. 地高辛(水溶性)(SERVA Electrophoresis GmbH,目录号:19551.02)
  3. 来自热解芽孢杆菌的热解菌素(Calbiochem ,目录号:58656)
  4. NaBr(Sigma-Aldrich,目录号:S-9756)
  5. NaSCN(Merck KGaA,目录号:6627)
  6. Na 2 CO 3(Merck KGaA,目录号:6392)
  7. NaOH(Roth North America,目录号:9097.2)
  8. 蔗糖(Roth North America,目录号:9097.2)
  9. EDTA(SERVA Electrophoresis GmbH,目录号:11280)
  10. HEPES(Roth North America,目录号:9105.3)

  11. (Roth North America,目录号:4984.3)。


  12. NaH R 2 PO 4(Roth North America,目录号:K300.2)
  13. 0.1 M磷酸钠缓冲液(见配方)
  14. 分馏缓冲液(参见配方)
  15. HS缓冲区(参见配方)
  16. 0.4%洋地黄素溶液(见配方)
  17. 在HS缓冲液中的嗜热菌蛋白酶储备溶液(40mg/ml)(参见配方)
  18. 含有HS缓冲液的盐(参见配方)

设备

  1. 离心机(Beckmann Coulter,型号:Avanti J-25; Eppendorf,型号:5430R)
  2. 超速离心机(Beckmann Coulter,型号:Optima LE-80K)
  3. Sonifier(Branson,型号:B-12)
  4. 光度计(Amersham biosciences,型号:Ultraspe TM 3100pro)

程序

  1. 类囊体膜的分馏
    1. 溶解类囊体在颗粒边缘混合5毫升新鲜 分离类囊体(0.8mg叶绿素/ml)   5ml 0.4%的洋地黄皂苷溶液,并在室温下孵育2分钟。 避免 通过轻微搅拌沉积类囊体溶液。
    2. 通过加入90ml冰冷的分馏缓冲液停止溶解。
    3. 将溶液离心15分钟(10,000×g,4℃)。 小心 保持上清液的下一步,并尽量避免污染 从丸。将颗粒(颗粒部分)溶解在2ml 分馏缓冲液并储存在冰上。
    4. 离心机 步骤A3的上清液30分钟(40,000×g,4℃)。小心取出 上部的上清液用于步骤A5并留下约3cm 该溶液避免基质层片段的污染。 (可选)将颗粒溶解在剩余的上清液中并保存  冰上的间歇馏分 颗粒基质层 将步骤A4的上清液超速离心60分钟(100,000× g,4℃)。小心地取出上清液并溶解沉淀 分级缓冲液。尽量保持浓度尽可能高 以避免在调整时的额外的超速离心步骤 分数至期望的叶绿素浓度。储存 天然类囊体级分(-20℃或-80℃)高叶绿素 建议浓度在2-4 mg/ml左右。用于SDS-PAGE分析 最终浓度从0.25至1μgChl /μl,取决于 目的蛋白。 作为一般规则,使用较低的最终蛋白质 浓度较大的膜蛋白与许多半胱氨酸减少 (例如 CP47,PsaA)。 较高的蛋白质浓度适合于低浓度 分子量蛋白质以减少样品体积并避免 扩展的加载缓冲区前面可能会干扰迁移 的非常小的蛋白质
    5. 在80%丙酮中测量级分的叶绿素含量,并调整到等量的μg叶绿素/μl。
    6. 通过SDS-PAGE分析分馏(图1)。

  2. 类囊体膜的盐处理
    1. 将新鲜分离的类囊体膜溶解在HS缓冲液(0.5mg叶绿素/ml)中和含有HS缓冲液的盐中。
    2. 在冰上孵育样品30分钟。
    3. 用两份HS缓冲液稀释样品。
    4. 通过离心10分钟(20,000×g,4℃)分离成沉淀和可溶性级分。
    5. 通过SDS-PAGE分析级分。

  3. 类囊体膜的嗜热菌蛋白酶处理
    1. 将新鲜分离的类囊体膜溶于HS缓冲液(0.5mg叶绿素/ml)中
    2. 产生50%的内向外和50%右侧外囊泡 超声波脉冲(10-30秒,10-30次)到冰上的溶液中。 等待 在用于冷却的脉冲之间约20秒。
    3. 添加蛋白酶   嗜热菌蛋白酶至终浓度为100μg/ml 类囊体膜溶液和内侧和右侧外囊泡
    4. 在不同的时间点(例如0,1,2,5分钟)取探针, 并立即通过加入EDTA终止消化 浓度为20mM。
    5. 在含有20mM EDTA的HS缓冲液中洗涤探针
    6. 通过SDS-PAGE比较类囊体和内侧和右侧外囊泡的消化。

代表数据



图1.类囊体系统


图2.分馏程序(A)的方案


图3.类囊体分离(A)的考马斯染色将类囊体膜(T)分成基质层(S),间歇部分(I)和颗粒层(G),分离的蛋白质随后用考马斯染色以判断级分的纯度。 PSI蛋白PsaA和PsaB以及ATP合酶亚基CFoα/β在基质层中高度富集,在颗粒膜中仅存在少量。 PSII蛋白CP47,CP43,D1,D2和PSII(LHCII)的光捕获复合物的天线蛋白主要存在于颗粒部分中,但几乎缺乏基质层片段。间歇部分含有两种光系统的蛋白质。加入等量的叶绿素(5μg)

图4.洗涤实验(B)。将用不同含盐缓冲液处理的类囊体膜分级成沉淀(P)和上清液(S),并通过SDS-PAGE分离。通过考马斯染色和免疫印迹分析分析分离的蛋白质。可以在所有使用的盐条件下通过考马斯染色来鉴定亲水性ATP合酶亚基CFoα/β从膜上的解离。相反,PSII(LHCII)和PSI核心亚基PsaA/B的疏水性光捕获复合物不能从沉淀部分释放。特异性抗血清用于鉴定与PSII相关的外周腔Psb0蛋白。只有在严格的盐条件下(2M NaSCN和0.1M NaOH),PsbO蛋白才能完全从膜上释放。


图5.蛋白酶处理方案(C)


图6.拓扑学研究(C)。 未处理和超声处理的类囊体与嗜热菌蛋白酶孵育,并使用PsbO和PsaE抗血清进行免疫处理。在未处理的类囊体中,管腔Psb0受到保护 来自嗜热菌蛋白酶处理,而基质暴露的PsaE降解。 在形成约50%内向外囊泡的超声处理的类囊体中,PsbO部分降解,而PsaE被部分保护。

笔记

  1. 为了尽可能快地和温和地溶解大的类囊体颗粒,使用精细的油漆刷
  2. 对于通过SDS-PAGE分离类囊体膜蛋白,尤其是≤10kDa的蛋白,强烈推荐将Tricine-SDS-PAGE用于参考(Schägger,2006)。
  3. 对于低分子量类囊体膜蛋白的免疫印迹分析,使用0.2μMPVDF或0.1μM硝酸纤维素膜并缩短转移时间。

食谱

  1. 0.1M磷酸钠缓冲液(pH7.4) 1 M HEPES-NaOH(pH 8.0)
    0.5 M EDTA(pH 8)
  2. 分馏缓冲液
    100mM蔗糖 10mM磷酸钠缓冲液(pH7.4) 5mM MgCl 2/
    5mM NaCl
  3. HS缓冲区
    0.1 M蔗糖 10mM HEPES-NaOH(pH8.0)
  4. 0.4%的洋地黄皂苷溶液
    通过混合和加热
    将40mg洋地黄皂苷溶解在10ml H 2 O中 溶液可以储存在-20°C
  5. 嗜热菌蛋白酶储备溶液(40mg/ml)在HS缓冲液中
    库存可以储存在-20°C
  6. 含有HS缓冲液的盐
    2 HSB缓冲液中的NaBr HS缓冲液中的2 M NaSCN
    在HS缓冲液中的0.1M Na CO 2 3 0.1mM NaOH的HS缓冲液A

致谢

该协议根据Karnauchov等人之前的工作(1997)改编或修改。这项研究得到了德国科学基金会(Deutsche Forschungsgemeinschaft(ME 1794)to J. M.

参考文献

  1. Armbruster,U.,Zuhlke,J.,Rengstl,B.,Kreller,R.,Makarenko,E.,Rühle,T.,Schünemann,D.,Jahns,P.,Weisshaar,B.,Nickelsen, Leister,D。(2010)。 拟南芥类囊体蛋白PAM68是有效的D1生物发生和光系统II所必需的 植物细胞 22(10):3439-3460。
  2. Dalcorso,G.,Pesaresi,P.,Masiero,S.,Aseeva,E.,Schünemann,D.,Finazzi,G.,Joliot,P.,Barbato,R.and Leister, 含有PGRL1和PGR5的复合物参与在线性和循环电子流之间的转换,拟南芥。 细胞 132(2):273-285。
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How to cite this protocol: Torabi, S., Plöchinger, M. and Meurer, J. (2014). Localization and Topology of Thylakoid Membrane Proteins in Land Plants. Bio-protocol 4(24): e1363. DOI: 10.21769/BioProtoc.1363; Full Text



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