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Candida albicans Mitochondrial Protein Import Assay
白念珠菌线粒体蛋白的转运实验   

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Abstract

We have established the use of Candida albicans as a new model system to study mitochondrial biogenesis. This dimorphic yeast provides an excellent system to investigate the coordination of mitochondrial biogenesis with other cellular networks including cellular metabolism and the cell cycle. Unlike the model lab yeast Saccharomyces cerevisiae, C. albicans is not subject to the Crabtree effect and hence grows aerobically in glucose when oxygen is present. Therefore the control of mitochondrial biogenesis in C. albicans is more typical of eukaryotic cells. C. albicans has a fully sequenced genome and there are many published tools for genetic manipulation facilitating Systems Biology approaches. The isolation of mitochondria (as described in the protocol: Preparation of Mitochondria from Candida albicans) (Hewitt et al., 2013) produces a more simplified system that can be interrogated using the standard tools of molecular biology. In addition, the import of radiolabelled proteins described in this protocol is a sensitive technique that can be used to determine details of kinetics and interactions of imported proteins.

Keywords: Mitochondria(线粒体), Yeast(酵母), Fungal pathogens(病原真菌), Protein targeting(蛋白质导向), Metabolism(代谢)

Materials and Reagents

  1. Rabbit Reticulocyte Lysate System, Nuclease Treated (Promega Corporation, catalog number: L4960 )
  2. 35S labeled cysteine and methionine (PerkinElmer, catalog number: 072007MC )
  3. SUPERase-In (Life Technologies, Ambion®, catalog number: AM2696
  4. Mitochondria
  5. Sorbitol
  6. Valinomycin
  7. Oligomycin
  8. Antimycin
  9. Soybean Trypsin Inhibitor (SBTI) (Sigma-Aldrich)
  10. Amino acids
  11. Coomassie Brilliant Blue G250
  12. Digitonin (Calbiochem, catalog number: CAS 11024-24-1 )
  13. RNA for in vitro translation (see Recipes)
  14. Import buffer (IB) (see Recipes)
  15. Blue native lysis buffer (see Recipes)
  16. 10x BN (Blue Native) loading dye (see Recipes)
  17. RNA (see Recipes)
  18. ATP (see Recipes)
  19. NADH (see Recipes)
  20. AVO (see Recipes)
  21. Proteinase K (PK) (see Recipes)
  22. Apyrase (see Recipes)
  23. Phenylmethanesulfonylfluoride (PMSF) (see Recipes)
  24. Trypsin (see Recipes)

Equipment

  1. Water bath with temperature control
  2. Centrifuge
  3. Filter – 0.45 μm Acrodisc syringe filter (Pall Life Sciences)

Procedure

  1. Translation Reaction (30 °C for 50 min) – 15 μl reaction, scale as needed.
    1. 10 μl rabbit reticulocyte lysate.
    2. 1 μl amino acids mix (1 mM but no cysteine or methionine).
    3. 1 μl RNA
    4. 1 μl 35S labeled cysteine and methionine
    5. 1 μl SUPERase-In (add a maximum of 2.5 μl).
  2. Mitochondria Preparation
    1. 100 μl of Import buffer (IB) per 50 μg of mitochondria
    2. Standard conditions for 100 μl IB:
      1. ATP (0.4 M) 1 μl
      2. NADH (0.5 M) 0.4 μl
      3. Mix by inversion and leave at import temperature (25 °C standard) for 5 min to equilibrate.
      4. Add 5 μl translation reaction per 100 μl import (reaction mix can be diluted by addition of water and 2.4 M sorbitol to a final sorbitol concentration of 0.6 M so up to 15 μl of this solution can be added to 100 μl of import buffer which can help increase signal if translation efficiency is low) and incubate at import temperature removing samples at suitable time points as described in part 3.
    3. Optional additions:
      1. AVO mix of mitochondrial inhibitors for dissipation of membrane potential (1 μl per 100 μl).
      2. Apyrase- 2 μl per 100 μl IB – treat lysate and mitochondria for 10 min at 25 °C.
  3. Stopper solutions
    1. 100 μl cold IB per 100 μl import in standard conditions
    2. Additions include the following but concentrations may need to be optimized:
      1. Trypsin: 1 μl per 100 μl of stopper solution
      2. SBTI: 4 μl per 200 μl of mix
      3. PK: 2 μl per 100 μl of stopper solution
      4. PMSF: 1 mM final (vortex when added)
      5. PK/trypsin containing solutions need 20 min before addition of inhibitor
    3. At each time point remove 95 μl if adding 5 μl translation reaction/100 μL IB or 105 μl if adding 15 μl/100 μl IB.
  4. Sample Treatment
    1. Once inhibitor added to all samples spin at 4 °C/8,000 x g/10 min.
    2. Remove and discard supernatant.
    3. Add 100 μl cold IB (do not resuspend pellet – add protease inhibitor if protease used in stopper solution).
    4. Spin at 4 °C/8,000 x g/5 min.
    5. Remove and discard supernatant.
      (For SDS resuspend pellet in appropriate amounts of water and SDS loading dye)
      For blue native PAGE:
    6. Resuspend pellet in 50 μl cold blue native lysis buffer (see Recipes) per 2 μl of mitochondria.
    7. Leave samples on ice for 15 min with gentle vortex every 5 min.
      (FOR ANTIBODY SHIFT: Add 2 μl antibody and leave on ice for 1 h – gentle vortex every 20 min – then continue as per method below)
    8. Spin samples at 4 °C/14,000 x g/10 min (or 15,000 if available).
    9. Remove 45 μl supernatant to new tube with 5 μl blue Native loading dye.

Recipes

  1. RNA from in vitro transcription reaction using 5 μg linear plasmid DNA (or 1.5 μg of PCR product with SP6 promoter)
  2. Import buffer (IB)
    0.6 M sorbitol
    50 mM HEPES (adjust to pH7.4 with KOH)
    2 mM KPi (pH 7.4 made by mixing 1 M KH2PO4 with 1 M K2HPO4)
    25 mM KCl
    10 mM MgCl2
    0.5 mM EDTA
    1 mM DTT stored at RT (room temperature)
  3. ATP
    Aliquots of 0.4 M stored at -20 °C (pH to 7)
  4. NADH
    Aliquots of 0.5 M stored at -20 °C
  5. AVO
    100x stock frozen in EtOH & foiled (1 μM valinomycin, 20 μM oligomycin, 8 μM antimycin) stored at -20 °C
  6. PK (Proteinase K)
    5 mg/ml aliquots in IB stored at -20 °C
  7. PMSF
    0.2 M in EtOH stored at RT for up to 1 month
  8. Trypsin
    Use 10 mg/ml stock (to give 0.1 mg/ml final) for 20 min (stored at -20 °C)
  9. SBTI
    50 mg/ml (for 5 min on ice) (stored at -20 °C)
  10. Apyrase
    500 units/ml in 50% glycerol stored at -20 °C
  11. 10x BN (Blue Native) loading dye
    0.5 M aminocaproic acid
    0.1 M Bis Tris pH 7.0
    10% Coomassie Brilliant Blue G250 – filter and store at room temperature
  12. Blue native lysis buffer
    20 mM Tris-HCl
    0.1 mM EDTA
    50 mM NaCl
    10% glycerol (store at room temperature)
    Add PMSF and 5% w/v digitonin (Calbiochem – heat to dissolve and store at 4 °C) to give a final concentration of 1% digitonin and 1 mM PMSF just before use.

Acknowledgments

The protocol was adapted from: Hewitt et al. (2012). The work in the Traven lab and Lithgow lab on Candida albicans mitochondria is supported by a project grant from the Australian National Health and Medical Research Council (APP1023973). V.H. is the recipient of an Australian Postgraduate Award.

References

  1. Chan, N. C. and T. Lithgow (2008). The peripheral membrane subunits of the SAM complex function codependently in mitochondrial outer membrane biogenesis. Mol Biol Cell 19(1): 126-136.
  2. Hewitt, V. L., Heinz, E., Shingu-Vazquez, M., Qu, Y., Jelicic, B., Lo, T. L., Beilharz, T. H., Dumsday, G., Gabriel, K., Traven, A. and Lithgow, T. (2012). A model system for mitochondrial biogenesis reveals evolutionary rewiring of protein import and membrane assembly pathways. Proc Natl Acad Sci U S A 109(49): E3358-3366.
  3. Hewitt, V., Lithgow, T. and Traven, A. (2013). Preparation of Mitochondria from Candida albicans. Bio-protocol 3(4): e330. 

简介

我们已经建立了白色念珠菌(Candida albicans)作为研究线粒体生物发生的新模式系统的用途。这种二聚酵母提供了一个优秀的系统来调查线粒体生物发生与其他细胞网络,包括细胞代谢和细胞周期的协调。与模拟实验室酵母酿酒酵母不同, 白色素不受Crabtree效应的影响,因此当存在氧时,在葡萄糖中有氧生长。因此在C中控制线粒体生物发生。白色念珠菌是更典型的真核细胞。 C。白色念珠菌具有完全测序的基因组,并且有许多公开的用于遗传操作促进系统生物学方法的工具。线粒体的分离(如方案中所述:从假丝酵母制备线粒体白蛋白 )(Hewitt等人,2013)产生更简化的系统,其可以使用分子生物学的标准工具来查询。此外,本协议中描述的放射性标记的蛋白质的输入是一种敏感的技术,可用于确定进口蛋白质的动力学和相互作用的细节。

关键字:线粒体, 酵母, 病原真菌, 蛋白质导向, 代谢

材料和试剂

  1. 兔网织红细胞溶解物系统,Nuclease Treatment(Promega Corporation,目录号:L4960)
  2. 标记的半胱氨酸和甲硫氨酸(PerkinElmer,目录号:072007MC)
  3. SUPERase-In(Life Technologies,Ambion ®,目录号:AM2696) 
  4. 线粒体
  5. 山梨醇
  6. 维拉霉素
  7. 寡霉素
  8. 抗霉素
  9. 大豆胰蛋白酶抑制剂(SBTI)(Sigma-Aldrich)
  10. 氨基酸
  11. 考马斯亮蓝G250
  12. Digitonin(Calbiochem,目录号:CAS 11024-24-1)
  13. RNA用于体外翻译(参见Recipes)
  14. 导入缓冲区(IB)(参见配方)
  15. 蓝色天然裂解缓冲液(见配方)
  16. 10x BN(蓝色本地)负载染料(参见配方)
  17. RNA(参见配方)
  18. ATP(参见配方)
  19. NADH(见配方)
  20. AVO(参见配方)
  21. 蛋白酶K(PK)(参见配方)
  22. Apyrase(参见配方)
  23. 苯基甲磺酰氟(PMSF)(参见配方)
  24. 胰蛋白酶(见配方)

设备

  1. 带温度控制的水浴
  2. 离心机
  3. 过滤器 - 0.45μmAcrodisc注射器过滤器(Pall Life Sciences)

程序

  1. 翻译反应(30℃,50分钟) - 15μl反应,根据需要标度。
    1. 10μl兔网织红细胞裂解液
    2. 1μl氨基酸混合物(1mM,但不含半胱氨酸或甲硫氨酸)
    3. 1μlRNA
    4. 1μl 35 S标记的半胱氨酸和甲硫氨酸
    5. 1μlSUPERase-In(最多添加2.5μl)。
  2. 线粒体制备
    1. 100微升导入缓冲液(IB)每50微克线粒体
    2. 100μlIB:
      的标准条件
      1. ATP(0.4M)1μl
      2. NADH(0.5M)0.4μl
      3. 通过倒置混合并在进口温度(25℃标准)下静置5分钟以平衡
      4. 每100μl导入加入5μl翻译反应(反应混合物可以通过加入水和2.4 M山梨醇稀释至0.6 M的最终山梨醇浓度,因此高达15μl的此溶液可以添加到100μl的进口缓冲液 如果转换效率低则帮助增加信号),并在进口温度下孵育,在合适的时间点除去样品,如第3部分所述
    3. 可选添加:
      1. AVO混合的线粒体抑制剂用于消耗膜电位(每100μl1μl)
      2. Apyrase-每100μlIB2μl - 在25℃处理裂解物和线粒体10分钟。
  3. 塞子解决方案
    1. 在标准条件下,每100μl进口加入100μl冷IB
    2. 添加包括以下内容,但可能需要优化浓度:
      1. 胰蛋白酶:每100μl终止溶液1μl/μl
      2. SBTI:4μl/200μl混合物
      3. PK:每100μl终止溶液2μl/μl
      4. PMSF:1mM终浓度(加入时为涡流)
      5. 含PK /胰蛋白酶的溶液在加入抑制剂之前需要20分钟
    3. 在每个时间点删除95 如果添加5微升翻译反应/100微升IB或105微升,如果加入15微升/100微升IB。
  4. 样品处理
    1. 一旦添加到所有样品中的抑制剂以4℃/8,000×g/10分钟旋转。
    2. 取出并弃去上清液。
    3. 加入100μl冷IB(不重悬沉淀 - 如果蛋白酶用于塞子溶液中,则加入蛋白酶抑制剂)
    4. 以4℃/8,000转/分钟旋转/5分钟。
    5. 取出并弃去上清液。
      ( 对于蓝色原生PAGE:
    6. 重悬在50μl冷蓝色天然裂解缓冲液(见Recipes)每2微升线粒体。
    7. 让样品在冰上15分钟,用温和的涡旋每5分钟 (抗体移位:加入2μl抗体,在冰上放置1小时 - 每20分钟轻轻涡旋一次,然后按照下面的方法继续)
    8. 以4℃/14,000×10 6 g/10分钟(或15,000,如果可得到的话)旋转样品。
    9. 删除45μl上清液到新管用5μl蓝色天然装载染料

食谱

  1. 来自使用5μg线性质粒DNA(或1.5μg具有SP6启动子的PCR产物)的体外转录反应的RNA。
  2. 导入缓冲区(IB)
    0.6M山梨醇 50mM HEPES(用KOH调节至pH7.4)
    2mM KPi(通过将1μMKH 2 PO 4与1MK 2 HPO 4混合而制备的pH 7.4)
    25 mM KCl
    10mM MgCl 2/
    0.5mM EDTA 在室温(室温)存储的1mM DTT
  3. ATP
    将0.4M的等分试样储存在-20℃(pH至7)下
  4. NADH
    0.5微升的等分试样储存在-20℃下
  5. AVO
    将100×储存在EtOH& (1μM缬氨霉素,20μM寡霉素,8μM抗霉素),保存在-20℃下
  6. PK(蛋白酶K)
    将5mg/ml等分试样储存在-20℃的IB中
  7. PMSF
    0.2M在EtOH中,在室温下储存最多1个月
  8. 胰蛋白酶
    使用10mg/ml原液(最终给予0.1mg/ml)20分钟(储存在-20℃)
  9. SBTI
    50mg/ml(在冰上5分钟)(储存在-20℃)
  10. Apyrase
    500单位/ml在-20℃下贮存的50%甘油中
  11. 10x BN(蓝色本机)装载染料
    0.5 M氨基己酸
    0.1 M Bis Tris pH 7.0
    10%考马斯亮蓝G250 - 过滤并在室温下贮存
  12. 蓝色天然裂解缓冲液
    20mM Tris-HCl
    0.1mM EDTA
    50mM NaCl 10%甘油(在室温下储存)
    加入PMSF和5%w/v毛地黄皂苷(Calbiochem-加热溶解并在4℃储存),使得在使用前最终浓度为1%洋地黄皂苷和1mM PMSF。

致谢

该方案改编自:Hewitt等人(2012)。在Traven实验室和Lithgow实验室在白色假丝酵母(Candida albicans)线粒体上的工作得到澳大利亚国家卫生和医学研究理事会(APP1023973)的项目赠款的支持。 V.H.是澳大利亚研究生奖的获得者。

参考文献

  1. Chan,N.C.和T.Lithgow(2008)。 SAM复合物功能的外周膜亚基在线粒体外膜生物发生中独立地。 Mol Biol Cell 19(1):126-136
  2. Hewitt,VL,Heinz,E.,Shingu-Vazquez,M.,Qu,Y.,Jelicic,B.,Lo,TL,Beilharz,TH,Dumsday,G.,Gabriel,K.,Traven,A.and Lithgow ,T.(2012)。 线粒体生物发生的模型系统揭示了蛋白质输入和膜组装途径的进化重新布线。 Proc Natl Acad Sci U S A 109(49):E3358-3366。
  3. Hewitt,V.,Lithgow,T。和Traven,A。(2013)。从线粒体的制备 3(4): e330。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hewitt, V. L., Lithgow, T. and Traven, A. (2013). Candida albicans Mitochondrial Protein Import Assay. Bio-protocol 3(4): e331. DOI: 10.21769/BioProtoc.331.
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