搜索

Preparation of Mitochondria from Candida albicans
白念珠菌的线粒体制备   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Based on the methods of (Daum et al., 1982) and (Hewitt et al., 2012), 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, which has been widely used in the mitochondrial biogenesis field, C. albicans is not subject to the Crabtree effect, 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 this protocol produces a more simplified system that can be interrogated using the standard tools of molecular biology. In addition the import of radiolabelled proteins as described in the protocol: Candida albicans Mitochondrial Protein Import Assay (Hewitt et al., 2013) is a sensitive technique that can be used to determine details of kinetics and interactions of imported proteins.

Materials and Reagents

  1. Candida albicans culture
  2. Tris-SO4
  3. DTT
  4. Sorbitol
  5. Lyticase (Sigma-Aldrich, catalog number: L2524 )
  6. K+MES [Potassium 2-(N-morpholino)ethanesulfonate]
  7. K+HEPES [Potassium 4-(2-hydroxyethyl)-1-piperazineethanesulfonate]
  8. PMSF
  9. Tris-DTT buffer (see Recipes)
  10. Sorbitol buffer (see Recipes)
  11. KH2PO4 & K2HPO4 (see Recipes)
  12. Breaking Buffer (BB) 6.0 (see Recipes)

Equipment

  1. Incubator shaker with temperature control
  2. Centrifuges
  3. Dounce (tight dounce 40 ml) (Wheaton, catalog number: 06-435C )

Procedure

  1. Culture preparation
    Inoculate with sufficient starter culture to produce a culture of Candida albicans cells with an OD600 = 1.0-2.0 (an OD600 of up to 7 has worked in our hands) at the desired start time the next morning. Shake at 200 rpm at 30 °C overnight.
  2. Method
    1. Collect cells by spinning at 4,000 x g/10 min/RT (room temperature).
    2. Pour off medium
    3. Resuspend cells in dH2O in weighed centrifuge tubes.
    4. Spin at 2,500 x g/5 min/RT. Reweigh to get mass of cells.
    5. Resuspend cells in Tris-DTT buffer (see Recipes) ~5 ml per g of cells.
    6. Incubate for 15 min at 30 °C with gentle shaking (~100 rpm).
    7. Spin at 2,500 x g/5 min/RT.
    8. Resuspend pellet in ~5 ml/g of cells pre-warmed 1.2 M sorbitol buffer.
    9. Spin at 2,500 x g/5 min/RT.
    10. Weigh out 0.2 mg/g of cells of lyticase. Dissolve in 2 ml pre-warmed (30 °C) 1.2 M sorbitol buffer per gram of cells. Resuspend pellet in this solution.
    11. Incubate cells in lyticase solution for ~60 min/30 °C with gentle shaking (~100 rpm) or until spheroplasts form (check spheroplast formation by osmotic shock: Add 30 μl cells to 2 ml 1.2 M sorbitol and water. After vortexing water sample should go clear).
    12. Spin down for 2,500 x g/5 min/RT. Discard supernatant.
    13. Resuspend in cold 1.2 M sorbitol buffer ~5 ml/g of cells. Spin 2,500 x g/5 min/4 °C.
    14. Resuspend in minimal amount of cold breaking buffer (BB) 6.0 then make up in BB 6.0 containing 1 mM PMSF final (~4 ml/g cells)
    15. Homogenize 15 times using a tight dounce. Dounce should be ~3/4 full or a bit less. Up stroke must be fast and steady. Bubbles can break mitochondria so try not to let it pop out.
    16. Spin homogenate 5 min/4 °C/3,000 x g. Save supernatant in a new tube and keep on ice (supernatant should be cloudy).
    17. (Optional step for higher yield) Resuspend pellet again in BB 6.0 with PMSF and tight dounce and spin as above. Combine supernatants. Discard pellets.
    18. Spin combined supernatants 5 min/4 °C/3,000 x g. Save supernatant (repeat this spin step to clear more of the contaminating membranes if still getting a large pellet).
    19. Spin supernatant for 10 min/4 °C/12,000 x g.
    20. Pour off the supernatant and resuspend pellet in a small amount of BB 7.4 (with bovine serum albumin if mitochondria are to be used for in vitro import assays).
    21. Spin down 10 min/4 °C/12,000 x g then remove any white membranes that surround reddish-brown mitochondrial pellet before resuspending in a minimal amount of BB 7.4 with BSA.
    22. Repeat spin step above if still contaminating membranes present.
    23. Estimate the mitochondrial concentration as follows: Add 10 μl crude mitochondria to 990 μl 0.6% SDS. As a blank add 10 μl BB 7.4 with BSA to 990 μl 0.6% SDS. Measure A280 using Quartz cuvette. An absorbance value of 0.21 corresponds to 10 mg/ml protein in the undiluted mixture.
    24. Aliquot at appropriate volumes and snap freeze in dry ice/liquid nitrogen.

Recipes

  1. Tris-DTT
    0.1 M Tris-SO4 (pH 9.4)
    10 mM DTT (make fresh from frozen 1 M DTT aliquots just before use)
  2. 1.2 M Sorbitol buffer
    1.2 M sorbitol
    20 mM KPi (pH 7.4)
  3. Breaking buffer, BB (6.0)
    0.6 M sorbitol
    20 mM K+MES (pH 6.0) (add PMSF just prior to use to 1 mM final concentration)
  4. PMSF
    34 mg/ml (0.2 M) in ethanol
  5. Breaking buffer, BB (7.4)
    0.6 M sorbitol
    20 mM K+HEPES (pH 7.4) (adjust pH with KOH)
  6. 2.4 M sorbitol stock (aqueous solution deionised)
  7. 1 M K+MES stock (pH 6.0)
    Filter sterilize and store in foil at RT
  8. 1 M KPi (pH 7.4)
    Make from stocks of 1 M KH2PO4 and stock of 1 M K2HPO4 and mix them until pH = 7.4. Do not titrate with HCl or NaOH.

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. Daum, G., P. C. Bohni, et al. (1982). Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem 257(21): 13028-13033.
  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). Candida albicans Mitochondrial Protein Import Assay. Bio-protocol 3(4): e331. 

简介

基于(Daum等人,1982)和(Hewitt等人,2012)的方法,我们已经建立了白色念珠菌的使用作为研究线粒体生物发生的新模型系统。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。不同于模型实验室酵母,酿酒酵母(Saccharomyces cerevisiae),其已经广泛用于线粒体生物发生领域,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。如本方案中所述的线粒体的分离产生更简化的系统,其可以使用分子生物学的标准工具来查询。此外,如方案中所述的放射性标记蛋白的导入: 白色念珠菌/em>线粒体蛋白质输入测定(Hewitt等人,2013)是一种敏感技术,其可用于确定进口蛋白质的动力学和相互作用的细节。

材料和试剂

  1. 白色念珠菌文化
  2. Tris-SO
  3. DTT
  4. 山梨醇
  5. 裂解酶(Sigma-Aldrich,目录号:L2524)
  6. K + MES [2-(N-吗啉代)乙磺酸钾]
  7. K] + HEPES [4-(2-羟乙基)-1-哌嗪乙磺酸钾]
  8. PMSF
  9. Tris-DTT缓冲液(见配方)
  10. 山梨醇缓冲液(见配方)
  11. KH sub 2 PO 4 4& K 2 HPO 4 (请参阅配方)
  12. 断裂缓冲液(BB)6.0(参见配方)

设备

  1. 带温度控制的培养箱摇床
  2. 离心机
  3. Dounce(浓杜松40毫升)(Wheaton,目录号:06-435C)

程序

  1. 文化准备
    用足够的起始培养物接种以产生具有OD 600 = 1.0-2.0(OD 600至多7)的白色念珠菌细胞的培养物 已经在我们手中)在第二天早上所需的开始时间。 在200rpm下在30℃摇动过夜。
  2. 方法
    1. 通过以4000转/分钟/em/10分钟/RT(室温)旋转收集细胞。
    2. 倒掉介质
    3. 重悬细胞在称重的离心管中的dH 2 O中。
    4. 以2500转/g/5分钟/RT旋转。 重新获得大量的细胞。
    5. 重悬细胞在Tris-DTT缓冲液(见配方)〜5毫升每克细胞
    6. 在30℃温和振荡(〜100rpm)孵育15分钟。
    7. 以2500转/g/5分钟/RT旋转。
    8. 在约5ml/g细胞预热的1.2M山梨醇缓冲液中重悬沉淀。
    9. 旋转2,500英寸x g /5分钟/RT
    10. 称取0.2mg/g裂解酶细胞。溶解于每克细胞2ml预热(30℃)1.2M山梨醇缓冲液中。在此溶液中重悬沉淀。
    11. 孵育细胞裂解酶溶液约60分钟/30℃,轻轻摇动(〜100 rpm)或直到原生质体形成(通过渗透休克检查原生质球形成:添加30微升细胞到2毫升1.2 M山梨醇和水涡旋水样品应清除)。
    12. 旋转2,500分钟/g/5分钟/RT。弃去上清液。
    13. 重悬于冷的1.2M山梨醇缓冲液〜5ml/g细胞。旋转2,500×g/5/5分钟/4℃。
    14. 在最少量的冷断裂缓冲液(BB)6.0中重悬,然后在含有1mM PMSF终浓度(〜4ml/g细胞)的BB6.0中补充。
    15. 使用紧密的dounce匀浆15次。 Dounce应该〜3/4满或少一点。上升行程必须快速稳定。气泡可以打破线粒体,所以尽量不要让它弹出
    16. 旋转匀浆5分钟/4℃/3,000×g /天。将上清液保存在新管中并保持在冰上(上清液应该是混浊的)。
    17. (用于更高产率的任选步骤)用PMSF和紧密的dounce并如上所述旋转在BB 6.0中再次悬浮颗粒。合并上清液。丢弃颗粒。
    18. 旋转混合的上清液5分钟/4℃/3,000×g。保存上清液(如果仍然得到大的沉淀,则重复该旋转步骤以清除更多的污染膜)。
    19. 旋转上清液10分钟/4℃/12,000xg。
    20. 倒出上清液并将沉淀重悬于少量BB 7.4(如果线粒体用于体外进口测定,则用牛血清白蛋白)。
    21. 然后旋转10分钟/4℃/12,000×g 然后除去任何围绕红棕色线粒体沉淀的白色膜,然后用少量的BB 7.4与BSA重悬浮。
    22. 如果仍存在污染膜,请重复上述步骤
    23. 估计线粒体浓度如下:添加10微升粗线粒体到990微升0.6%SDS。 作为空白添加10μlBB 7.4与BSA至990μl0.6%SDS。 使用Quartz比色杯测量A <280>。 0.21的吸光度值对应于未稀释的混合物中的10mg/ml蛋白质。
    24. 等分适量,在干冰/液氮中快速冷冻

食谱

  1. Tris-DTT
    0.1M Tris-SO 4(pH 9.4)
    10mM DTT(在即将使用前从冷冻的1M DTT等分试样中制成新鲜)
  2. 1.2 M山梨醇缓冲液
    1.2M山梨醇 20mM KPi(pH7.4)
  3. 断开缓冲区,BB(6.0)
    0.6M山梨醇 20mM K on + MES(pH 6.0)(在使用前加入PMSF至1mM终浓度)
  4. PMSF
    34mg/ml(0.2M)的乙醇溶液
  5. 断开缓冲区,BB(7.4)
    0.6M山梨醇 20mM K on + HEPES(pH 7.4)(用KOH调节pH)
  6. 2.4M山梨醇原液(去离子水溶液)
  7. 1 M K + MES原液(pH 6.0)
    过滤灭菌,并在室温下保存在金属箔
  8. 1 M KPi(pH 7.4)
    由1M KH sub 2 PO 4和1 MK 2 sub HPO 4的储备物制备并混合,直至 pH = 7.4。 不要用HCl或NaOH滴定。

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)线粒体上的工作由澳大利亚国家卫生和医学研究委员会(APP1023973)的项目资助支持。 V.H.是澳大利亚研究生奖的获得者。

参考文献

  1. Daum,G.,P.C.Bohni,et al。 (1982)。 将蛋白质导入线粒体。细胞色素b2和细胞色素c过氧化物酶位于酵母线粒体的膜间隙中。 J Biol Chem 257(21):13028-13033。
  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)。 白色念珠菌线粒体蛋白输入测定。 生物协议 3(4):e331。 
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hewitt, V. L., Lithgow, T. and Traven, A. (2013). Preparation of Mitochondria from Candida albicans. Bio-protocol 3(4): e330. DOI: 10.21769/BioProtoc.330.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。