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Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells
HeLa细胞中线粒体内外膜的分离   

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PeerJ
May 2014

Abstract

Mitochondria are organelles that have important functions in oxidative phosphorylation, fatty acid oxidation and apoptosis signaling. They have two distinct membranes, outer membrane (OM) and inner membrane (IM). IM contains respiratory chain complexes that produce ATP. IM is rich in cardiolipin, a specific phospholipid reportedly having a critical role for organizing super-complex formation of respiratory chain complexes. IM abundant in cardiolipin exhibits resistance to extraction by digitonin (a non-ionic detergent), whereas the detergent easily lyses OM. Therefore, digitonin is useful to separate mitoplast (IM plus matrix) and OM from mitochondria. Here, we describe a method to isolate mitochondria from HeLa cells, and a method to isolate mitochondrial outer membrane proteins and inner membrane proteins by using digitonin. This method is applicable also to other types of cultured cells such as COS-7.

Keywords: Mitochondria (线粒体), Membrane (膜), Isolation (隔离), Inner (内部的), Outer (外面的)

Materials and Reagents

  1. HeLa cells (80-100% confluent) pre-cultured in 8 to 12 pieces of dishes (diameter 10-cm)
  2. PBS (-) (without Ca2+ and Mg2+)
  3. EDTA
  4. Mannitol
  5. Sucrose
  6. HEPES
  7. EGTA
  8. Complete Mini EDTA-free (Roche Diagnostics, catalog number: 11 836 170 001 )
  9. Digitonin (Wako Pure Chemical Industries, catalog number: 040-02123 )
  10. Trichloroacetic acid (optional)
  11. Mitochondria isolation buffer (MTiso-buffer) (see Recipes)
  12. Digitonin stock solution (see Recipes)
  13. Digitonin buffer (see Recipes)

Equipment

  1. Culture dishes
  2. Aspirator
  3. Cell scraper (e.g. Rubber policeman)
  4. Dounce homogenizer (Glass 7 ml Dounce Tissue Grinder) (WHEATON, catalog number: 357542 )
  5. 50 ml-tube
  6. Phase-contrast microscope (upright-type, x100 ~ x400) (for confirming disruption efficiency of the cells)
  7. Centrifuges [for centrifugation at 500 x g (swing rotor) and 10,000 x g (angle rotor)]
  8. Micro tube mixer (e.g. TOMY MT-400 , Digital Biology, catalog number: MT-400) or vortex mixer
  9. Ultracentrifuge (e.g. Hitachi, catalog number: CS100 ) (for centrifugation at 100,000 x g (angle rotor)

Procedure

  1. Remove medium from the culture dishes (harboring 80-100% confluent cells) by aspiration, and then add 3-4 ml of PBS (-) containing 1 mM EDTA into each dish.
  2. After 5 min, scrape the cells by using a cell scraper (or by gentle pipetting), collect the cells into a 50 ml-tube, and centrifuge at 500 x g (5 min, 4 °C). Then, remove the supernatant by aspiration.
  3. Re-suspend the pelleted cells with 50 ml of PBS (-) by inverting the tube, and then centrifuge it at 500 x g (5 min, 4 °C), followed by removing the supernatant by aspiration.
  4. Suspend the pelleted cells with 5 ml of MTiso-buffer by inverting the tube, and then transfer the suspension into Dounce homogenizer pre-cooled on ice.
  5. Homogenize the cell suspension by dounce homogenization [about 50 strokes (up and down)]. If necessary, verify the disruption efficiency of the cells under microscope.
  6. Pile up the homogenate on an equal volume of 340 mM sucrose in a 50 ml-tube, and centrifuge it at 500 x g (10 min, 4 °C) to remove nuclei and unbroken cells as pellet.
  7. Collect the supernatant in another tube (e.g. 5 ml-tube or some pieces of 1.5 ml-tubes), and then centrifuge it at 10,000 x g (10 min, 4 °C) to isolate mitochondria as pellet.
  8. Re-suspend the isolated mitochondria with 1 ml of digitonin buffer containing appropriate concentration of digitonin (usually 0.15-5 mg/ml, see Recipes) (digitonin extraction).
  9. Mix the suspension intensely for 15 min by using micro tube mixer (at max speed) or vortex mixer. If necessary, add an equal volume of MTiso-buffer to stop digitonin extraction.
  10. Centrifuge the suspension at 10,000 x g (10 min, 4 °C) to isolate the pellet containing mitoplast (IM plus matrix) and the supernatant containing solubilized OM and inter-membrane space (IMS) proteins.
  11. To separate IM fraction and matrix fraction, re-suspend the pellet (mitoplast) in small amount of MTiso-buffer (usually 0.1-0.5 ml), gently sonicate it to disrupt mitoplast in the water-bath-type sonicator filled with ice-cold water, and then centrifuge it at 100,000 x g (30 min, 4 °C), giving IM fraction as pellet and matrix fraction as supernatant.
  12. (Optional) If it is necessary to concentrate soluble proteins, please perform TCA-precipitation method using trichloroacetic acid.

Recipes

  1. Mitochondria isolation buffer (MTiso-buffer)
    3 mM HEPES-KOH (pH 7.4)
    210 mM mannitol
    70 mM sucrose
    0.2 mM EGTA
    Complete Mini EDTA-free (protease inhibitor cocktail)
  2. Digitonin stock solution (freshly prepared)
    4% digitonin in water (heating at 60 °C will improve the insolubility of digitonin)
  3. Digitonin buffer (MTiso-buffer containing 0.15-5 mg/ml digitonin)
    Prepare the buffer by mixing MTiso-buffer and Digitonin stock solution
    Note: The efficiency of digitonin extraction largely depends on the quality of the digitonin reagent (e.g. manufacture and lot number). It is recommended to test the efficiency at several different concentration of digitonin. Digitonin extraction at appropriate concentration will clearly separate OM and mitoplast, although the treatment at higher concentration will solubilize not only OM but also IM.

References

  1. Nishimura, N., Gotoh, T., Oike, Y. and Yano, M. (2014). TMEM65 is a mitochondrial inner-membrane protein. PeerJ 2: e349.
  2. Pallotti, F. and Lenaz, G. (2007). Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol 80: 3-44.
  3. Schnaitman, C. and Greenawalt, J. W. (1968). Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell Biol 38(1): 158-175.
  4. Yano, M., Hoogenraad, N., Terada, K. and Mori, M. (2000). Identification and functional analysis of human Tom22 for protein import into mitochondria. Mol Cell Biol 20(19): 7205-7213.
  5. Yano, M., Kanazawa, M., Terada, K., Namchai, C., Yamaizumi, M., Hanson, B., Hoogenraad, N. and Mori, M. (1997). Visualization of mitochondrial protein import in cultured mammalian cells with green fluorescent protein and effects of overexpression of the human import receptor Tom20. J Biol Chem 272(13): 8459-8465.

简介

线粒体是在氧化磷酸化,脂肪酸氧化和凋亡信号传导中具有重要功能的细胞器。 它们有两个不同的膜,外膜(OM)和内膜(IM)。 IM含有产生ATP的呼吸链复合物。 IM富含心磷脂,据报道,特定磷脂具有组织呼吸链复合物的超复合物形成的关键作用。 富含心磷脂的IM表现出对毛地黄皂苷(非离子型洗涤剂)的提取的抗性,而洗涤剂容易裂解OM。 因此,毛地黄皂苷可用于将线粒体(IM +基质)和OM与线粒体分离。 在这里,我们描述了一种从Hela细胞分离线粒体的方法,以及通过使用毛地黄皂苷分离线粒体外膜蛋白和内膜蛋白的方法。 该方法也适用于其它类型的培养细胞如COS-7。

关键字:线粒体, 膜, 隔离, 内部的, 外面的

材料和试剂

  1. 在8至12个培养皿(直径10-cm)中预培养的HeLa细胞(80-100%汇合)
  2. PBS( - )(不含Ca 2+ 2+和Mg 2+ 2 + )
  3. EDTA
  4. 甘露醇
  5. 蔗糖
  6. HEPES
  7. EGTA
  8. Complete Complete无EDTA(Roche Diagnostics,目录号:11 836 170 001)
  9. digitonin(Wako Pure Chemical Industries,目录号:040-02123)
  10. 三氯乙酸(可选)
  11. 线粒体分离缓冲液(MTiso缓冲液)(参见配方)
  12. 去甲肾上腺素储备溶液(见配方)
  13. digitonin缓冲液(见配方)

设备

  1. 文化菜肴
  2. 吸气器
  3. 细胞刮刀(如橡胶警察)
  4. Dounce匀浆器(玻璃7ml Dounce Tissue Grinder)(WHEATON,目录号:357542)
  5. 50 ml管
  6. 相差显微镜(直立型,x100〜x400)(用于确认细胞的破坏效率)
  7. 离心机[用于在500×g(摇摆转子)和10,000×g(转子转子)上离心]
  8. 微管混合器(例如TOMY MT-400,Digital Biology,目录号:MT-400)或涡流混合器
  9. 超速离心机(例如,Hitachi,目录号:CS100)(用于在100,000×g (角转子)离心)

程序

  1. 通过抽吸从培养皿(包含80-100%融合细胞)中除去培养基,然后向每个培养皿中加入3-4ml含有1mM EDTA的PBS( - )。
  2. 5分钟后,通过使用细胞刮擦器(或通过温和移液)刮擦细胞,将细胞收集到50ml管中,并以500×g离心(5分钟,4℃)。然后,通过抽吸除去上清液。
  3. 通过翻转管,用50ml PBS( - )重悬浮沉淀的细胞,然后以500×g离心(5分钟,4℃),随后通过抽吸除去上清液。
  4. 通过翻转管将沉淀的细胞用5ml MTiso缓冲液悬浮,然后将悬浮液转移到在冰上预冷却的Dounce匀浆器中。
  5. 通过dounce匀浆[约50次(上下)]均质化细胞悬浮液。如果需要,验证细胞在显微镜下的破坏效率。
  6. 将匀浆在等体积的340mM蔗糖中在50ml管中堆积,并以500×g(10分钟,4℃)离心以除去作为沉淀的核和未破碎的细胞。
  7. 在另一个管(例如5ml管或一些1.5ml管)中收集上清液,然后以10,000×g离心(10分钟,4℃) )分离线粒体作为沉淀。
  8. 用1ml含适当浓度的毛地黄皂苷(通常为0.15-5mg/ml,参见Recipes)(毛地黄皂苷提取物)的毛地黄皂苷缓冲液重悬浮分离的线粒体。
  9. 通过使用微管混合器(以最大速度)或涡流混合器强烈混合悬浮液15分钟。如有必要,加入等体积的MTiso缓冲液以终止洋地黄皂苷的提取。
  10. 在10,000×g(10分钟,4℃)离心悬浮液以分离含有丝裂原体(IM +基质)和含有增溶的OM和膜间空间(IMS)蛋白的上清液。
  11. 为了分离IM部分和基质部分,将沉淀物(间质体)重悬浮于少量MTiso缓冲液(通常为0.1-0.5ml)中,轻轻超声处理以在装有冰冷的水浴型超声发生器中破坏叶绿体水,然后以100,000×g(30分钟,4℃)离心,得到作为沉淀的IM级分和作为上清液的基质级分。
  12. (可选)如果需要浓缩可溶性蛋白质,请使用三氯乙酸进行TCA沉淀法。

食谱

  1. 线粒体分离缓冲液(MTiso缓冲液)
    3mM HEPES-KOH(pH7.4) 210mM甘露糖 70mM蔗糖 0.2 mM EGTA
    完全迷你无EDTA(蛋白酶抑制剂混合物)
  2. 降血脂素储备溶液(新鲜制备)
    4%洋地黄皂甙在水中(在60℃加热将改善毛地黄皂苷的不溶性)
  3. 去离子蛋白缓冲液(含有0.15-5mg/ml毛地黄皂苷的MTiso缓冲液)
    通过混合MTiso缓冲液和digitonin储备液制备缓冲液
    注意:毛地黄皂苷提取的效率主要取决于毛地黄皂苷试剂的质量(例如制造和批号)。建议测试几种不同浓度的毛地黄皂苷的效率。在适当浓度下的降钙素提取将清楚地分离OM和丝裂原体,尽管在较高浓度下的治疗不仅将溶解OM而且也溶解IM。

参考文献

  1. Nishimura,N.,Gotoh,T.,Oike,Y.and Yano,M.(2014)。 TMEM65是一种线粒体内膜蛋白。 PeerJ 2:e349。
  2. Pallotti,F。和Lenaz,G。(2007)。 线粒体从动物细胞和组织培养系中的分离和亚分级。方法Cell Biol 80:3-44。
  3. Schnaitman,C。和Greenawalt,J.W。(1968)。 大鼠肝线粒体内膜和外膜的酶性质。 Cell Biol 38(1):158-175
  4. Yano,M.,Hoogenraad,N.,Terada,K.and Mori,M。(2000)。 人类Tom22蛋白质进入线粒体的鉴定和功能分析 Cell Biol 20(19):7205-7213
  5. Yano,M.,Kanazawa,M.,Terada,K.,Namchai,C.,Yamaizumi,M.,Hanson,B.,Hoogenraad,N.and Mori,M.(1997)。 在具有绿色荧光蛋白的培养的哺乳动物细胞中线粒体蛋白输入的可视化和人类输入的过表达的作用 受体Tom20.J J Biol Chem 272(13):8459-8465。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nishimura, N. and Yano, M. (2014). Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells. Bio-protocol 4(22): e1299. DOI: 10.21769/BioProtoc.1299.
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Ligia Fao
Center for Neuroscience and cell biology
I could not obtain sample from matrix and outer membrane. The only band that I could detect doing WB it was anti-complexII in the inter membrane sample.
10/2/2018 4:30:21 PM Reply