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Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-3H]dihydrosphingosine
使用放射性D-赤氏-[4,5-3氢]二氢神经鞘氨醇对酵母鞘脂类进行代谢标记   

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参见作者原研究论文

本实验方案简略版
Molecular Microbiology
Dec 2012

Abstract

Yeast sphingolipids can be metabolically labeled with exogenously added radioactive precursors. Here we describe a method to label ceramides and complex sphingolipids of Saccharomyces cerevisiae with a radioactive ceramide precursor, D-erythro-[4, 5-3H]dihydrosphingosine. This protocol is used to study the biosynthesis, transport and metabolism of sphingolipids in yeast.

Keywords: Sphingolipids (鞘脂), Yeast (酵母), Metabolism (代谢), Radioactive precusor (放射性前体), Dihydrosphingosine (二氢)

Materials and Reagents

  1. Yeast strain (Saccharomyces cerevisiae)
  2. D-erythro-[4,5-3H]-dihydrosphingosine ([3H]DHS) (60 Ci/mmol) (American Radiolabeled Chemicals, catalog number: ART-46 )
  3. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S1504 )
  4. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 )
  5. Glass beads (0.5 mm) (Yasui Kikai, catalog number: YGB05 )
  6. Chloroform (Sigma-Aldrich, catalog number: 05-3400 )
  7. Methanol (Sigma-Aldrich, catalog number: 19-2410 )
  8. 1-Butanol (Sigma-Aldrich, catalog number: 03-4560 )
  9. Ammonium hydroxide (Sigma-Aldrich, catalog number: 221228 )
  10. Acetic acid (Sigma-Aldrich, catalog number: 01-0280 )
  11. Thin layer chromatography silica gel 60 (TLC plate) (aluminium sheet 20 x 20 cm ) (Merck KGaA, catalog number: 105553 )
  12. N2 gas
  13. Distilled water (H2O)
  14. Dextrose (D-glucose) (Sigma-Aldrich, catalog number: 07-0680 )
  15. Yeast extract (Oriental Yeast, catalog number: OYC42008000 )
  16. Polypeptone (Wako Chemicals USA, catalog number: 513-76611 )
  17. Yeast nitrogen base without amino acids and ammonium sulfate (BD DifcoTM, catalog number: 233520 )
  18. Adenine (Sigma-Aldrich, catalog number: A9126 )
  19. Leucine (Nacalai Tesque, catalog number: 20327-62 )
  20. Histidine (Nacalai Tesque, catalog number: 18116-92 )
  21. Lysine (Sigma-Aldrich, catalog number: L5626 )
  22. Uracil (Nacalai Tesque, catalog number: 35824-82 )
  23. Ammonium sulfate (Sigma-Aldrich, catalog number: A2939 )
  24. Tryptophan (Nacalai tesque, catalog number: 35607-32 )
  25. Yeast extract-peptone dextrose (YPD) liquid medium (see Recipes)
  26. Synthetic minimal dextrose (SD) liquid medium (see Recipes)
  27. Water-saturated 1-butanol (see Recipes)

Equipment

  1. Tritium-sensitive imaging plate (Fujifilm, catalog number: BAS-TR2040 )
  2. Microcentrifuge tube
  3. Shaking incubator (TAITEC, model: BR-43FL.MR )
  4. Swinging bucket centrifuge (TOMY, model: LC-201 )
  5. Microcentrifuge (TOMY, model: MX-301 )
  6. Water bath shaker (TAITEC, model: PERSONAL-11 )
  7. Bath-type ultrasonic cleaner (AS ONE, model: US-2R )
  8. Pressure gas blowing concentrator (EYELA, model: MGS-2200 )
  9. TLC developing tank
  10. Hair dryer
  11. FLA-7000 imaging system (GE Healthcare Life Sciences, model: Typhoon FLA 7000 )

Software

  1. FLA-7000 image software (ImageQuant TL analysis)

Procedure

  1. Cell culture and metabolic labeling
    1. Inoculate yeast cells in YPD liquid medium at 25 °C with gyratory shaking at 175-200 rpm overnight.
    2. Dilute the culture with SD liquid medium to an OD600 of 0.01-0.02 and culture with gyratory shaking at 175-200 rpm overnight.
    3. When the OD600 of the culture is 0.2-0.6, transfer the culture to a 50 ml conical tube.
    4. Spin down yeast cells by a swinging bucket centrifuge at 2,000 x g for 5 min at room temperature (RT) and remove supernatant.
    5. Resuspend the pellet in 20 ml SD liquid medium, spin down at 2,000 x g for 5 min at RT and remove supernatant. Repeat this step at least three times.
    6. Resuspend cells in SD liquid medium to get an OD600 of 20 and transfer 0.5 ml of cell suspension to a new 50 ml conical tube.
    7. Incubate at 25 °C for 20 min with reciprocal shaking.
    8. Add 4 μCi of [3H] DHS to the cell culture and incubate at 25 °C for 1-4 h with reciprocal shaking. If necessary, lipid synthesis inhibitors are added before the labeling.

  2. Lipid extraction and alkaline hydrolysis
    1. To stop metabolic labeling, chill the 50 ml conical tube on ice and add 250 mM NaF and 250 mM NaN3 to a final concentration of 10 mM.
    2. Transfer the labeled culture to a 1.5 ml microcentrifuge tube (tube #1).
    3. Collect yeast cells by a microcentrifuge at 20,000 x g for 5 min at 4 °C, remove supernatant and resuspend yeast cells in 1 ml cold water. Repeat this step at least three times.
    4. Adjust volume of cell suspension to 66 μl with cold water and vortex well.
    5. Add 0.3 g of glass beads and vortex well at low temperature (vortex for 30 sec and chill on ice for 1-2 min, repeat at least three times).
    6. Add 440 μl of chloroform-methanol (CM; 1/1, v/v), vortex well and centrifuge at 20,000 x g for 5 min at RT.
    7. Transfer the supernatant to a new 1.5 ml microcentrifuge tube (tube #2).
    8. In order to extract the radiolabeled lipids remaining in tube #1, add 200 μl of chloroform-methanol-water (CMW; 10/10/3, v/v/v) in tube #1, sonicate for approximately 5-10 min in bath-type ultrasonic cleaner until the pellet is completely suspended, centrifuge at 20,000 x g for 5 min at RT and transfer the supernatant to tube #2.
    9. Dry the combined supernatants in tube #2 completely with N2 gas using pressure gas blowing concentrator.
    10. Add 80 μl of CMW, vortex well and centrifuge at 20,000 x g for 1 min at RT.
    11. To deacylate glycerophospholipids by mild alkaline hydrolysis, add 16 μl of 0.6 N NaOH in methanol, vortex well and incubate at 30 °C for 3 h.
    12. Centrifuge at 20,000 x g for 1 min at RT.
    13. To neutralize, add 16 μl of 0.6 N acetic acid in methanol, vortex well and centrifuge at 20,000 x g for 1 min at RT.
    14. Dry the reaction mixture completely with N2 gas using pressure gas blowing concentrator.
    15. To desalt, add 100 μl of water to tube #2, vortex well and spin down at 20,000 x g for 1 min at RT.
    16. Further add 200 μl of water-saturated 1-butanol, vortex well, centrifuge 20,000 x g for 5 min at RT and transfer the butanol (upper) phase containing sphingolipids to a new 1.5 ml microcentrifuge tube (tube #3).
    17. In order to collect the radiolabeled sphingolipids remaining in tube #2, add 200 μl of water-saturated 1-butanol to tube #2, vortex well, centrifuge 20,000 x g for 5 min at RT and transfer the butanol phase to tube #3. Repeat this step one more time.
    18. Add 100 μl of water to the combined butanol phase in tube #3, vortex well, centrifuge 20,000 x g for 5 min at room temperature and transfer the butanol phase to a new 1.5 ml microcentrifuge tube (tube #4).
    19. Dry the butanol phase in tube #4 completely with N2 gas using pressure gas blowing concentrator.

  3. Lipid separation and analysis
    1. Add 25 μl of CMW to tube #4, vortex well and centrifuge at 20,000 x g for 1 min at RT.
    2. Then load total sample on TLC plate.
    3. Place the plate in a glass TLC developing tank and develop with chloroform-methanol-4.2 N ammonium hydroxide (9/7/2, v/v/v) solvent mixture.
    4. When wetting front reaches within 1 cm of the top of TLC plate, remove the plate from the tank and dry it at RT.
    5. When the plate is completely dried with a hair dryer, set it in exposure cassette and expose it to a tritium-sensitive imaging plate for few hours-few days.
    6. Capture image and quantify signals with FLA-7000 image analyzer and software (Figure 1A).

  4. Ceramide extraction and analysis
    1. Add a few drops of water on the area containing ceramides of TLC plate, which is in 2 or 3 cm inside from the top edge of the plate.
    2. Collect the silica of the area by scraping with a spatula and transfer to a 1.5 ml microcentrifuge tube (tube #5).
    3. Add 400 μl of CM, sonicate for approximately 5 min in bath-type ultrasonic cleaner until the silica is completely suspended, centrifuge at 20,000 x g for 5 min at RT and transfer the supernatant to a new 1.5 ml microcentrifuge tube (tube #6).
    4. Add 200 μl of CM to tube #5, vortex well, centrifuge at 20,000 x g for 5 min at RT and transfer the supernatant to tube #6.
    5. Dry the combined supernatants in tube #6 completely with N2 gas using pressure gas blowing concentrator.
    6. Add 100 μl of water to tube #6, vortex well and spin down at 20,000 x g for 1 min at RT.
    7. Further add 200 μl of water-saturated 1-butanol, vortex well, centrifuge 20,000 x g for 5 min at RT and transfer the butanol phase containing ceramides to a new 1.5 ml microcentrifuge tube (tube #7).
    8. In order to collect the radiolabeled ceramides remaining in tube #6, add 200 μl of water-saturated 1-butanol to tube #6, vortex well, centrifuge 20,000 x g for 5 min at RT and transfer the butanol phase to tube #7. Repeat this step one more time.
    9. Dry the butanol phase in tube #7 completely with N2 gas usng pressure gas blowing concentrator.
    10. Add 25 μl of CMW to tube #7, vortex well and centrifuge at 20,000 x g for 1 min at RT.
    11. Then load total sample on TLC plate.
    12. Place the plate in a glass TLC developing tank and develop with chloroform-methanol-acetic acid (190/9/1, v/v/v) solvent mixture.
    13. When wetting front reaches within 1 cm of the top of TLC plate, remove the plate from the tank and dry it at RT.
    14. When the plate is completely dried with a hair dryer, set it in exposure cassette and expose it to a tritium-sensitive imaging plate for few hours-few days.
    15. Capture image and quantify signals with FLA-7000 image analyzer and software (Figure 1B).


      Figure 1. Captured images showing the distribution of complex sphingolipids (A) and ceramides (B) on TLC plates and quantified data. Wild-type cells were labeled with [3H]DHS at 25 °C for 3 h in the absence or presence of 2 μg/ml aureobasidin A (AbA), a specific inositol phosphorylceramide (IPC) synthase inhibitor (Funato and Riezman, 2001; Kajiwara et al., 2012). The labeled lipids were extracted, subjected to mild alkaline hydrolysis and analyzed by TLC with chloroform-methanol-4.2 N ammonium hydroxide (9/7/2, v/v/v) solvent mixture (A). Fractions containing ceramides in (A) (Funato and Riezman, 2001) were collected by scraping, and the radiolabeled ceramides were extracted from the silica and analyzed by TLC with chloroform-methanol-acetic acid (190/9/1, v/v/v) solvent mixture (B). Incorporation of [3H]DHS into complex sphingolipids (IPC-A,-B,-C,-D, MIPCs and M(IP)2Cs) and into ceramides (Cer-A, -B and -C) was quantified and determined as percentage of the total radioactivity in (A). IPC-A,-B,-C,-D and Cer-A,-B,-C are different IPC subclasses and ceramide species, respectively (Haak et al., 1997). The complex sphingolipids and ceramides can be identified by using mutants defective in the biosynthesis of specific sphingolipid species (Haak et al., 1997), by chemical treatment of isolated radiolabeled lipids (Puoti et al., 1991; Funato and Riezman, 2001) or by using the lipid standards that are isolated from [3H] inositol labeled sphingolipids (Puoti et al., 1991; Haak et al., 1997). MIPC, mannosyl inositolphosphorylceramide; M(IP)2C, mannosyl di(inositolphosphoryl)ceramide.

Recipes

  1. YPD liquid medium
    20 g Dextrose
    10 g Yeast extract
    20 g Polypeptone
    40 mg Uracil
    40 mg Adenine
    40 mg Tryptophan
    Add H2O to 1 L
    Sterilize by autoclaving.
  2. SD liquid medium
    20 g Dextrose
    1.7 g Yeast nitrogen base without amino acids and ammonium sulfate
    5.0 g Ammonium sulfate
    80 mg Uracil
    80 mg Adenine
    80 mg Leucine
    80 mg Histidine
    80 mg Lysine
    Dissolve in 800 ml H2O
    Adjust pH to 5.8-6.0 with 1 M NaOH
    Adjust volume to 990 ml with H2O
    Sterilize by autoclaving
    Add 10 ml of tryptophan solution (8 mg/ml) sterilized by filtration.
  3. Water-saturated 1-butanol
    Mix equal volumes of 1-butanol and H2O
    Shake overnight at RT and leave
    The resulting upper phase is water-saturated 1-butanol.

Acknowledgments

This protocol was adapted from Kajiwara et al. (2012). This work was supported by a grant from the Graduate School of Biosphere Science (Hiroshima University) to Kajiwara K. and by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to Funato K.

References

  1. Funato, K. and Riezman, H. (2001). Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast. J Cell Biol 155(6): 949-959.
  2. Haak, D., Gable, K., Beeler, T. and Dunn, T. (1997). Hydroxylation of Saccharomyces cerevisiae ceramides requires Sur2p and Scs7p. J Biol Chem 272(47): 29704-29710.
  3. Kajiwara, K., Muneoka, T., Watanabe, Y., Karashima, T., Kitagaki, H. and Funato, K. (2012). Perturbation of sphingolipid metabolism induces endoplasmic reticulum stress-mediated mitochondrial apoptosis in budding yeast. Mol  Microbiol  86(5): 1246-1261.
  4. Puoti, A., Desponds, C. and Conzelmann, A. (1991). Biosynthesis of mannosylinositolphosphoceramide in Saccharomyces cerevisiae is dependent on genes controlling the flow of secretory vesicles from the endoplasmic reticulum to the Golgi. J Cell Biol 113(3): 515-525.

简介

酵母鞘脂可以用外源添加的放射性前体代谢标记。 在这里我们描述了用放射性神经酰胺前体D-赤式 - [4,5] 3 H]二氢鞘氨醇标记神经酰胺和酿酒酵母的复杂神经鞘脂的方法。 该方案用于研究酵母中鞘脂的生物合成,转运和代谢。

关键字:鞘脂, 酵母, 代谢, 放射性前体, 二氢

材料和试剂

  1. 酵母菌株(酿酒酵母)
  2. D-赤藓 - [4,5-,3 H] - 二氢鞘氨醇([3 H] DHS)(60Ci/mmol)(American Radiolabeled Chemicals,目录号:ART -46)
  3. 氟化钠(NaF)(Sigma-Aldrich,目录号:S1504)
  4. 叠氮化钠(NaN 3)(Sigma-Aldrich,目录号:S2002)
  5. 玻璃珠(0.5mm)(Yasui Kikai,目录号:YGB05)
  6. 氯仿(Sigma-Aldrich,目录号:05-3400)
  7. 甲醇(Sigma-Aldrich,目录号:19-2410)
  8. 1-丁醇(Sigma-Aldrich,目录号:03-4560)
  9. 氢氧化铵(Sigma-Aldrich,目录号:221228)
  10. 乙酸(Sigma-Aldrich,目录号:01-0280)
  11. 薄层色谱法硅胶60(TLC板)(铝板20×20cm)(Merck KGaA,目录号:105553)
  12. N <2>气体
  13. 蒸馏水(H 2 O)
  14. 葡萄糖(D-葡萄糖)(Sigma-Aldrich,目录号:07-0680)
  15. 酵母提取物(Oriental Yeast,目录号:OYC42008000)
  16. 聚胨(Wako Chemicals USA,目录号:513-76611)
  17. 无氨基酸和硫酸铵的酵母氮源(BD Difco TM ,目录号:233520)
  18. 腺嘌呤(Sigma-Aldrich,目录号:A9126)
  19. 亮氨酸(Nacalai Tesque,目录号:20327-62)
  20. 组氨酸(Nacalai Tesque,目录号:18116-92)
  21. 赖氨酸(Sigma-Aldrich,目录号:L5626)
  22. 尿嘧啶(Nacalai Tesque,目录号:35824-82)
  23. 硫酸铵(Sigma-Aldrich,目录号:A2939)
  24. 色氨酸(Nacalai tesque,目录号:35607-32)
  25. 酵母提取物 - 蛋白胨葡萄糖(YPD)液体培养基(参见配方)
  26. 合成最小葡萄糖(SD)液体培养基(见配方)
  27. 水饱和的1-丁醇(参见配方)

设备

  1. 氚敏感成像板(Fujifilm,目录号:BAS-TR2040)
  2. 微量离心管
  3. 摇动培养箱(TAITEC,型号:BR-43FL MR)
  4. 摇摆式离心机(TOMY,型号:LC-201)
  5. 微量离心机(TOMY,型号:MX-301)
  6. 水浴摇动器(TAITEC,型号:PERSONAL-11)
  7. 浴式超声波清洗机(AS ONE,型号:US-2R)
  8. 压力气体吹浓缩器(EYELA,型号:MGS-2200)
  9. TLC显影槽
  10. 吹风机
  11. FLA-7000成像系统(GE Healthcare Life Sciences,型号:Typhoon FLA 7000)

软件

  1. FLA-7000图像软件(ImageQuant TL分析)

程序

  1. 细胞培养和代谢标记
    1. 在25℃下在YPD液体培养基中接种酵母细胞,在175-200rpm下回转振荡过夜
    2. 用SD液体培养基稀释培养物至0.01-0.02的OD 600,并在175-200rpm下回转振荡培养过夜。
    3. 当培养物的OD <600>为0.2-0.6时,将培养物转移到50ml锥形管中。
    4. 在室温(RT)下通过摇摆式离心机在2,000×g下旋转酵母细胞5分钟并除去上清液。
    5. 将沉淀重悬于20ml SD液体培养基中,在室温下以2,000xg离心5分钟,除去上清液。 重复此步骤至少三次。
    6. 将细胞悬浮在SD液体培养基中,使OD 600达到20,并将0.5ml细胞悬浮液转移到新的50ml锥形管中。
    7. 在25℃下伴随摇动孵育20分钟
    8. 向细胞培养物中加入4μCi的[3 H] DHS,并在25℃下伴随振荡孵育1-4小时。 如果需要,在标记之前加入脂质合成抑制剂。

  2. 脂质提取和碱水解
    1. 为了停止代谢标记,在冰上冷却50ml锥形管,并加入250mM NaF和250mM NaN 3至终浓度为10mM。
    2. 将标记的培养物转移到1.5ml微量离心管(管#1)
    3. 通过微量离心机在4℃下以20,000×g收集酵母细胞5分钟,除去上清液并将酵母细胞重悬于1ml冷水中。 重复此步骤至少三次。
    4. 用冷水将细胞悬液的体积调节至66μl,并涡旋振荡
    5. 加入0.3g玻璃珠,低温涡旋(涡旋30秒,在冰上冷却1-2分钟,重复至少三次)。
    6. 加入440μl氯仿 - 甲醇(CM; 1/1,v/v),涡旋并在室温下以20,000xg离心5分钟。
    7. 将上清液转移到新的1.5ml微量离心管(管#2)
    8. 为了提取管#1中剩余的放射性标记的脂质,在管#1中加入200μl氯仿 - 甲醇 - 水(CMW; 10/10/3,v/v/v),在超声处理中超声处理约5-10分钟浴式超声波清洗器,直到沉淀物完全悬浮,在室温下以20,000×g离心5分钟,并将上清液转移到#2管。
    9. 用压力气体吹扫浓缩器用N 2气完全干燥管#2中的合并的上清液。
    10. 加入80μlCMW,涡旋,并在室温下以20,000×g离心1分钟。
    11. 要通过温和的碱性水解去酰化甘油磷脂,加入16μl0.6N NaOH的甲醇溶液,涡旋混匀,在30℃孵育3小时。
    12. 在室温下以20,000×g离心1分钟。
    13. 为了中和,加入16μl0.6N乙酸的甲醇溶液,涡旋,并在室温下以20,000×g离心1分钟。
    14. 使用压力气体吹扫浓缩器用N 2气体完全干燥反应混合物
    15. 为了脱盐,向管#2中加入100μl水,涡旋,并在室温下以20,000×g离心1分钟。
    16. 进一步加入200μl水饱和的1-丁醇,涡旋,在室温下离心20,000×g离心5分钟,并将含有鞘脂的丁醇(上)相转移到新的1.5ml微量离心管(管# 3)。
    17. 为了收集保留在管#2中的放射性标记的鞘脂,向管#2中加入200μl水饱和的1-丁醇,涡旋,在室温下离心20,000xg离心5分钟,并转移丁醇相到管#3。重复此步骤一次。
    18. 向管#3中的合并的丁醇相中加入100μl水,涡旋,在室温下离心20,000×g离心5分钟,并将丁醇相转移到新的1.5ml微量离心管(#4管)。
    19. 使用压力气体吹扫浓缩器,用N 2气体完全干燥管#4中的丁醇相。

  3. 脂质分离和分析
    1. 向管#4中加入25μlCMW,涡旋,并在室温下以20,000×g离心1分钟。
    2. 然后将总样品加载到TLC板上
    3. 将板放在玻璃TLC显影槽中,用氯仿 - 甲醇-4.2N氢氧化铵(9/7/2,v/v/v)溶剂混合物显影。
    4. 当润湿前沿到达TLC板顶部1厘米以内时,从储罐中取出该板,并在室温下干燥
    5. 当板用吹风机完全干燥时,将其置于曝光盒中,并将其暴露于氚敏感成像板几小时 - 几天。
    6. 使用FLA-7000图像分析仪和软件捕获图像和量化信号(图1A)
  4. 神经酰胺提取和分析
    1. 在含有TLC板的神经酰胺的区域添加几滴水,该板位于板的顶部边缘内部2或3cm内。
    2. 通过用刮刀刮擦收集该区域的二氧化硅,并转移至1.5ml微量离心管(管#5)。
    3. 加入400μlCM,在浴型超声波清洗器中超声约5分钟,直到二氧化硅完全悬浮,在室温下以20,000×g离心5分钟,并将上清液转移到新的1.5ml微量离心机管(管#6)。
    4. 向管#5中加入200μlCM,涡旋,在室温下以20,000×g离心5分钟,并将上清液转移到管#6。
    5. 使用压力气体吹扫浓缩器,用N 2气体完全干燥管#6中的合并的上清液。
    6. 向管#6中加入100μl水,涡旋并在室温下以20,000×g离心1分钟。
    7. 进一步加入200μl水饱和的1-丁醇,涡旋,在室温下离心20,000×g离心5分钟,并将含有神经酰胺的丁醇相转移到新的1.5ml微量离心管(#7管)中。
    8. 为了收集保留在管#6中的放射性标记的神经酰胺,向管#6中加入200μl水饱和的1-丁醇,涡旋,在室温下离心20,000分钟5分钟, 丁醇相至管#7。 重复此步骤一次。
    9. 使用N 2气体吹扫压缩气体吹浓缩器完全干燥管#7中的丁醇相。
    10. 向管#7中加入25μlCMW,涡旋,并在室温下以20,000×g离心1分钟。
    11. 然后将总样品加载到TLC板上
    12. 将板放在玻璃TLC显影槽中,用氯仿 - 甲醇 - 乙酸(190/9/1,v/v/v)溶剂混合物显影。
    13. 当润湿前沿到达TLC板顶部1厘米以内时,从储罐中取出该板,并在室温下干燥
    14. 当板用吹风机完全干燥时,将其置于曝光盒中,并将其暴露于氚敏感成像板几小时 - 几天。
    15. 使用FLA-7000图像分析仪和软件捕获图像和量化信号(图1B)。


      图1.捕获的图像显示了在TLC板上的复杂神经鞘脂(A)和神经酰胺(B)的分布和定量数据。野生型细胞用[3 H] ]在25℃下在不存在或存在2μg/ml金黄担子素A(AbA),特异性肌醇磷酰甘油酰胺(IPC)合酶抑制剂(Funato和Riezman,2001; Kajiwara等人, em>,2012)。提取标记的脂质,进行温和的碱性水解,并用氯仿 - 甲醇-4.2N氢氧化铵(9/7/2,v/v/v)溶剂混合物(A)通过TLC分析。通过刮取收集(A)中含有神经酰胺的级分(Funato和Riezman,2001),从二氧化硅中提取放射性标记的神经酰胺,并用氯仿 - 甲醇 - 乙酸(190/9/1,v/v/v)溶剂混合物(B)。将[3 H] DHS掺入复杂鞘脂(IPC-A,-B,-C,-D,MIPC和M(IP)2 C 8)中并掺入神经酰胺(Cer-A,-B和-C)被定量并确定为(A)中总放射性的百分比。 IPC-A,-B,-C,-D和Cer-A,-B,-C分别是不同的IPC亚类和神经酰胺物种(Haak等人,1997)。复杂的神经鞘脂和神经酰胺可以通过使用在特定鞘脂类物质的生物合成中有缺陷的突变体(Haak等人,1997),通过化学处理分离的放射性标记的脂质来鉴定(Puoti等人, ,1991; Funato和Riezman,2001)或通过使用从[肌醇]标记的鞘脂中分离的脂质标准(Puoti等人, 1991; Haak等人,1997)。 MIPC,甘露糖基肌醇磷酰甘油酰胺; M(IP)2 C,甘露糖基二(肌醇磷酰)神经酰胺。

食谱

  1. YPD液体介质
    20克葡萄糖
    10g酵母提取物
    20克聚蛋白胨 40mg尿嘧啶
    40 mg腺嘌呤
    40 mg色氨酸
    将H <2> O添加到1 L
    通过高压灭菌消毒。
  2. SD液体介质
    20克葡萄糖
    1.7g不含氨基酸和硫酸铵的酵母氮源 5.0g硫酸铵
    80mg尿嘧啶
    80 mg腺嘌呤
    80mg亮氨酸
    80 mg组氨酸
    80mg赖氨酸
    溶解在800ml H 2 O中 用1M NaOH将pH调节至5.8-6.0 使用H 2 O调节体积至990ml
    高压灭菌
    灭菌 加入10ml通过过滤灭菌的色氨酸溶液(8mg/ml)
  3. 水饱和的1-丁醇
    将等体积的1-丁醇和H 2 O混合 在室温下摇动一夜,然后离开
    所得上相是水饱和的1-丁醇。

致谢

该协议改编自Kajiwara等人(2012)。这项工作是由生物圈科学研究生院(广岛大学),Kajiwara K.和日本科学促进会科学研究助理和教育,文化,体育,科学和技术的日本对Funato K.

参考文献

  1. Funato,K。和Riezman,H。(2001)。 酵母中神经酰胺从ER到高尔基体的水泡和非泡输送。 > J Cell Biol 155(6):949-959
  2. Haak,D.,Gable,K.,Beeler,T.and Dunn,T。(1997)。 酿酒酵母的羟基化神经酰胺需要Sur2p和Scs7p。 J Biol Chem 272(47):29704-29710。
  3. Kajiwara,K.,Muneoka,T.,Watanabe,Y.,Karashima,T.,Kitagaki,H.and Funato,K。(2012)。 神经鞘脂代谢紊乱会诱导内质网 网状应激介导的线粒体凋亡在芽殖酵母中。 Mol微生物 86(5):1246-1261。
  4. Puoti,A.,Desponds,C.and Conzelmann,A。(1991)。甘露糖基肌醇磷酸酰胺的生物合成 在酿酒酵母中依赖于控制分泌囊泡从内质网到高尔基体的流动。 J Cell Biol 113(3):515-525 。
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引用:Karashima, T., Kajiwara, K. and Funato, K. (2013). Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-3H]dihydrosphingosine. Bio-protocol 3(16): e862. DOI: 10.21769/BioProtoc.862.
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