Uptake Assay for Radiolabeled Peptides in Yeast

引用 收藏 提问与回复 分享您的反馈 Cited by



Eukaryotic Cell
Oct 2007



We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.


Di/tripeptides and larger oligopeptides are sources of amino acids for protein synthesis, or may serve as carbon and nitrogen precursors for energy production and biosynthesis of metabolites in all organisms. Uptake of di/tripeptides and oligopeptides across the cell membrane is facilitated by peptide transporters. Measuring the accumulation of radiolabeled peptide provides an experimental approach to determine uptake across a specific peptide transport protein in a variety of cell types, including the yeast Saccharomyces cerevisiae. Appropriate experimental design allows for the determination of kinetic parameters such as the affinity and capacity of system under investigation. Dipeptides, such as radiolabeled Leu-Leu, can be used to measure transport across the di/tripeptide transporter Ptr2p of S. cerevisiae. We use the transport of dipeptides across Ptr2p in yeast as an example.

Materials and Reagents

  1. Culture tube, up to 17 x 100 mm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-956-6B )
  2. 1.5 ml microfuge tubes
  3. HAWP membrane filter (EMD Millipore, catalog number: HAWP02400 )
  4. Saccharomyces cerevisiae (S. cerevisiae): such as W303 strain (MATa or MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) (ATCC, catalog number: 208352 )
    Note: Other strains can be used, although the growth medium will have to be adjusted if specific auxotrophic requirements are needed.
  5. Proline (Sigma-Aldrich, catalog number: P5607 )
  6. Yeast nitrogen base without (NH4)2SO4 and amino acids (BD, catalog number: 233510 )
  7. D-glucose (Sigma-Aldrich, catalog number: G7021 )
  8. Uracil (Sigma-Aldrich, catalog number: U1128 )
  9. Adenine hemisulfate salt (Sigma-Aldrich, catalog number: A9126 )
  10. Histidine-HCl (Sigma-Aldrich, catalog number: H-8000 )
  11. Leucine (Sigma-Aldrich, catalog number: L8000 )
  12. Tryptophan (Sigma-Aldrich, catalog number: T0254 )
  13. Radiolabeled dipeptide [3H]Leu-Leu (Custom synthesized)
  14. Leu-Leu (Sigma-Aldrich, catalog number: L-2752 )
  15. Minimal Proline (MP) broth (see Recipes)
  16. MP+ broth (see Recipes)
  17. 20% glucose (see Recipes)
  18. 2% glucose (see Recipes)
  19. 2x uptake medium (see Recipes)


  1. 250 ml flask
  2. Vortex mixer (VWR, catalog number: 97043-562 )
  3. Water bath (VWR, catalog number: 89501-476 )
  4. Microscope
  5. Incubator (set up at 30 °C) containing a rotator mixer (Thermo Fisher ScientificTM, Thermo Scientific model: 1640Q )
  6. Shaker incubator, 30 °C (Eppendorf, New Brunswick Scientific, model: C-25 )
  7. Hemocytometer
  8. Dry bath, 30 °C with blocks to hold microfuge 1.5 ml tubes (Thermo Fisher Scientific, catalog number: 88870002 )
  9. Manifold vacuum filtration cell harvester (EMD Millipore, catalog number: xx2702550 )
  10. Filter forceps (EMD Millipore, catalog number: XX6200006P )
  11. Liquid Scintillation vial, 6 ml Omni-vial with cap (Wheaton, catalog number: 225414 )
  12. Liquid Scintillation Cocktail (MP Biomedical CytoScint, catalog number: 882453 )
  13. Liquid Scintillation Counter, TRI-CARB 2000TR (PerkinElmer, catalog number: B291000 )


  1. Preparation of a working cell stock
    Yeast cells are grown in a culture tube containing 5 ml of minimal medium supplemented with proline as the nitrogen (N) source (MP) and compounds to satisfy the auxotrophic requirements (MP+ broth, see Recipes below) at 30 °C overnight in a rotator until a turbid culture is obtained. The minimal medium described in this protocol is designed for the W303 strain. Proline is added as a poor N source in order to induce the synthesis of the peptide uptake system of S. cerevisiae. Other minimal media can be used for strains of S. cerevisiae with different auxotrophic requirements.
  2. 1 ml of the above overnight culture is seeded into a 250 ml flask containing 50 ml of fresh MP+ broth and grown to log phase, corresponding to a density of approximately 5 x 106 cells/ml, taking about 4 h.
  3. Log phase cells are harvested by centrifugation (1,000 x g) and washed three times with a solution of 2% glucose to remove culture medium residue and maintain the cells in an energized state.
  4. Adjustment of the cell number
    The cell pellet is resuspended in 0.5 ml 2% glucose and the suspended cells are counted on a hemocytometer. Based on the hemocytometer results, a cell suspension is prepared at a final concentration of 1 x 108 cells/ml in 2% glucose.
  5. Setting-up the cell uptake
    The 2x uptake medium is warmed to 30 °C. 60 μl of cell suspension is dispensed into each of ten, 1.5 ml microfuge tubes. The tubes are transferred into a 30 °C dry bath block and incubated for 5 min. To begin the assay, 60 μl of pre-warmed, 2x uptake medium are added to the pre-warmed 60 μl yeast cell suspensions and the suspension carefully mixed by pipetting. The tubes are incubated for various time intervals. Generally, times should range from a minimum of 10 sec to a maximum of 10 min. For each uptake time, triplicate determinations are made. To control for non-specific association of the radiolabeled peptide with the cells, a negative control in which no peptide should be transported must be completed. This can be done by repeating the entire uptake procedure at 0 °C, when cells are not metabolically active. Alternatively, completing the uptake assay at 30 °C using an isogenic strain known to be lacking the transporter of interest can also serve as a negative control.
  6. Stopping the uptake
    Upon completion of the uptake interval, cells are harvested and washed by filtration on any manifold system such as the Manifold Vacuum Filtration Cell Harvester (see Equipment list). A membrane filter pre-wet in water (one filter is needed for each of the ten samples) is placed onto the filtration apparatus, the apparatus is assembled and the vacuum is turned on. To stop the uptake, 100 μl of the uptake/cell suspension mixtures are pipetted onto the center of the filter. Each filter is then washed 4 times with 1 ml of ice-cold water.
    1. The HAWP membrane filters needed to complete the assay are placed into a Petri dish with water to pre-wet. Filters are removed as needed and placed onto the filtration apparatus just prior to stopping the uptake assay.
    2. It is important to avoid loading the cell suspension near the rim of the filter as this could result in loss of cells if the filtration apparatus does not seal tightly around the filter.
  7. Radioactivity measurement
    Turn off the vacuum to the filtration device and disassemble to access the filters. Remove the filters with the retained yeast cells using filter forceps and place each into a separate liquid scintillation vial. One milliliter of scintillation fluid is added to each vial, which is capped and then shaken by hand to ensure that the filter is completely submerged in the cocktail. The vials are allowed to sit at room temperature for a minimum of one hour to allow for equilibration with the scintillation fluid and radioactivity is measured using a Liquid Scintillation Counter, per manufacturer’s instructions.

Data analysis

The uptake of the radioactive substrate is plotted in terms of nmol of substrate/109 cells vs. time. For each uptake time, triplicate determinations are made, averaged, and the mean ± standard deviation are plotted. To determine the specific uptake, subtract the negative control values from the total uptake. A representative graph for the uptake of radiolabeled Leu-Leu is presented below (Figure 1).

Figure 1. Uptake vs. time. Uptake of radiolabeled dileucine was determined for yeast cells at 30 °C (Total Uptake) and at 0 °C (Negative Control) and normalized to reflect uptake in nmol per 109 yeast cells. The specific uptake, indicated in red, is determined by subtracting the negative control from the total.


  1. The experiment described above measures accumulation, or uptake, of the substrate into the cell. If kinetic measurements of transport are to be made, then it is essential that the intracellular fate of the substrate under study is understood. For example, should the peptide be rapidly hydrolyzed during transport or immediately upon entering the cytoplasm, the determination of kinetics by the method described above is not valid. It is also essential to determine that the substrate is not metabolized extracellularly before transport as well. If extracellular hydrolysis occurs, then that substrate would not be suitable for uptake measurements because uptake of the radioactivity would be measuring amino acid uptake. Therefore, extraction and analysis of the extracellular medium and the intracellular contents after transport measurement to determine the physical state of the substrate is essential for kinetic transport studies, whereas if only assessing ‘uptake’ is the goal of the studies, then such determination is not necessary.
  2. This experiment may be repeated with various amounts of non-metabolized radioactive substrate to determine the kinetics of uptake by way of Michaelis-Menten type analysis. Uptake is measured at a time in the linear phase of accumulation as determined by measurement of uptake vs. time (Figure 1) at various substrate concentrations. For each uptake point, triplicate determinations were made, averaged, and the mean ± standard deviation are plotted. The plot of substrate concentration (S) vs. uptake (V) will yield the apparent Km and the apparent Vmax of the transport system under study. Vmax represents the maximum rate achieved by the system and the Km is the substrate concentration at which the uptake rate is half of the maximum. A representative analysis of transport kinetics is provided below (Figure 2).

    Figure 2. Kinetics of peptide transport. Uptake (V) was determined as a function of [3H]Leu-Leu concentration (S) at 30 °C (Total Uptake) and at 0 °C (Negative Control). Values were normalized to reflect uptake in nmol per 109 yeast cells. The specific uptake, indicated in red, is determined by subtracting the negative control from the total. Data were fitted by non-linear regression to determine the Km (118 ± 18 nM) and the predicted Vmax (82 ± 4 nmol/109 cells/min) values for the specific uptake component.


  1. Minimal Proline (MP) broth (1L)
    20 g glucose
    1.7 g yeast nitrogen base (YNB) without (NH4)2SO4 and amino acids
    1 g of proline as a nitrogen source.
    The glucose is dissolved in 900 ml water and autoclave-sterilized
    Once cooled, 100 ml of a filter sterilized 10x YNB-proline stock (17 g YNB and 10 g proline in 100 ml water) is added
    Note: Proline is used as a nitrogen source to induce the synthesis of the yeast peptide transport system.
  2. MP+ broth
    100 ml of MP broth
    1.0 ml of uracil (2 mg/ml in double distilled water, filter-sterilized)
    1.0 ml adenine sulfate (2 mg/ml 0.1 N HCl, filter-sterilized)
    0.2 ml of histidine (10 mg/ml, filter-sterilized)
    0.3 ml of leucine (10 mg/ml, filter-sterilized)
    0.2 ml of tryptophan (10 mg/ml 0.1 N HCl, filter-sterilized)
    Note: The above amino acids and nucleobases are added to satisfy the auxotrophic requirement of S. cerevisiae W303. The specific growth conditions may be modified for the yeast strain or toxic compound to be assayed.
  3. 20% glucose (100 ml)
    Per 100 ml contains 20 g glucose
    Dissolve the glucose in double distilled water
    Adjust the final volume to 100 ml and autoclave or filter-sterilize the solution
  4. 2% glucose
    Dilute the 20% glucose solution 1:10 in water
  5. 2x uptake medium
    This will be diluted 1:1 with the cell mixture to begin the uptake assay
    The 2x medium consists of 2% glucose and 20 mM sodium citrate-potassium phosphate (pH 5.5)
    This is supplemented with radiolabeled peptide [2 µCi/ml (14C) Leu-Leu] and non-radiolabeled peptide (150 µM Leu-Leu)
    Note: The amount of substrate (radiolabeled and non-radiolabeled peptide) included in the 2x uptake medium will vary based on the nature of the experiment (Uptake vs. Time or Uptake vs. Substrate Concentration) and must be determined empirically for each cell type. The values given above are a starting point.


This protocol was adapted from our previous studies (Cai et al., 2007; Cai et al., 2006). This work was supported by grants from the National Institute of General Medical Sciences GM-22087 and GM-46520.


  1. Cai, H., Kauffman, S., Naider, F. and Becker, J. M. (2006). Genomewide screen reveals a wide regulatory network for di/tripeptide utilization in Saccharomyces cerevisiae. Genetics 172(3): 1459-1476.
  2. Cai, H., Hauser, M., Naider, F. and Becker, J. M. (2007). Differential regulation and substrate preferences in two peptide transporters of Saccharomyces cerevisiae. Eukaryot Cell 6(10): 1805-1813.



[背景] 二肽/三肽是所有生物体氮,碳和氨基酸的主要来源之一。含有毒性氨基酸残基的合成肽提供了测定酿酒酵母中肽转运和/或利用的实验方法。通过细胞内肽酶或蛋白酶水解内化肽释放毒性残留物,导致在培养皿中铺板的细胞培养板上生长停滞的容易检测的区域(晕轮)。例如,在细胞内水解时,毒性肽Ala-Eth释放乙硫氨酸(Eth),甲硫氨酸拮抗剂干扰氨基酸掺入蛋白质中以及DNA和其它甲基化途径的正常甲基化,从而导致细胞死亡。当点样到酵母细胞的草坪上时,转运的二肽Ala-Eth将抑制生长,并且在含有Eth的有毒肽被点样的区域周围的细胞的细胞壁中形成清晰的"晕"(图1A)。本文描述的用于测定肽中毒性的测定。 (1)可以通过简单地使用推定的有毒化合物代替含有毒性氨基酸的肽来修饰以确定任何底物的毒性,或者(2)其可以被修饰以通过替换S来确定底物对任何微生物的毒性。酿酒酵母在用靶生物的测定中。它是一种简单,廉价和相对快速的方法,用于确定针对特定生物体和所测定的毒性部分修改的底物毒性。...


  1. 培养管,高达17×100mm(Thermo Fisher Scientific,Fisher Scientific,目录号:14-956-6B)
  2. 1.5 ml微量离心管
  3. HAWP膜过滤器(EMD Millipore,目录号:HAWP02400)
  4. 酿酒酵母(酿酒酵母):例如W303菌株(MATa或MATαleu2-3,112trp1-1 can1-100 ura3-1 ade2-1 his3- 11,15)(ATCC,目录号:208352)
  5. 脯氨酸(Sigma-Aldrich,目录号:P5607)
  6. 没有(NH 4)2 SO 2 SO 4和氨基酸(BD,目录号:233510)的酵母氮碱
  7. D-葡萄糖(Sigma-Aldrich,目录号:G7021)
  8. 尿嘧啶(Sigma-Aldrich,目录号:U1128)
  9. 腺嘌呤半硫酸盐(Sigma-Aldrich,目录号:A9126)
  10. 组氨酸-HCl(Sigma-Aldrich,目录号:H-8000)
  11. 亮氨酸(Sigma-Aldrich,目录号:L8000)
  12. 色氨酸(Sigma-Aldrich,目录号:T0254)
  13. 放射性标记的二肽[3 H] Leu-Leu(定制合成)
  14. Leu-Leu(Sigma-Aldrich,目录号:L-2752)
  15. 最小脯氨酸(MP)肉汤(见配方)
  16. MP +肉汤(见配方)
  17. 20%葡萄糖(见配方)
  18. 2%葡萄糖(见配方)
  19. 2x摄取培养基(见配方)


  1. 250 ml烧瓶
  2. 涡旋混合器(VWR,目录号:97043-562)
  3. 水浴(VWR,目录号:89501-476)
  4. 显微镜
  5. 包含旋转混合器(Thermo Fisher Scientific TM,Thermo Scientific型号:1640Q)的孵育器(设置在30℃)
  6. Shaker孵育器,30℃(Eppendorf,New Brunswick Scientific,型号:C-25)
  7. 血细胞计数器
  8. 干浴,30℃,保持微量离心管1.5ml管(Thermo Fisher Scientific,目录号:88870002),
  9. 歧管真空过滤细胞收集器(EMD Millipore,目录号:xx2702550)
  10. 过滤钳(EMD Millipore,目录号:XX6200006P)
  11. 液体闪烁小瓶,6ml带盖的全方位小瓶(Wheaton,目录号:225414)
  12. 液体闪烁混合物(MP Biomedical CytoScint,目录号:882453)
  13. 液体闪烁计数器,TRI-CARB 2000TR(PerkinElmer,目录号:B291000)


  1. 制备工作细胞库
    酵母细胞在含有5ml作为氮(N)源(MP)的脯氨酸和满足营养缺陷要求的化合物(MP +肉汤,参见下文)的培养管中在30℃下在转子中培养过夜生长直到获得混浊培养物。该方案中描述的基本培养基是为W303菌株设计的。加入脯氨酸作为贫N源,以诱导S的肽摄取系统的合成。酿酒厂。其他基本培养基可用于S菌株。具有不同营养缺陷需求的酿酒酵母。
  2. 将1ml上述过夜培养物接种到含有50ml新鲜MP +肉汤的250ml烧瓶中,并生长至对数期,对应于约5×10 6个细胞/ml的密度,取大约4小时。
  3. 通过离心(1000×g)收集对数期细胞,并用2%葡萄糖溶液洗涤3次以除去培养基残余物并保持细胞处于通电状态。
  4. 调整单元格号
    将细胞沉淀重悬于0.5ml 2%葡萄糖中,并在血细胞计数器上计数悬浮的细胞。基于血细胞计数器的结果,制备在2%葡萄糖中的终浓度为1×10 8个细胞/ml的细胞悬浮液。
  5. 设置细胞摄取量
  6. 停止吸收
    在摄取间隔完成后,收获细胞并通过在任何歧管系统例如Manifold Vacuum Filtration Cell Harvester上过滤洗涤(参见设备列表)。将在水中预湿的膜过滤器(十个样品中的每一个需要一个过滤器)放置在过滤装置上,组装装置并打开真空。为了停止摄取,将100μl吸收/细胞悬浮液混合物吸移到过滤器的中心。然后将每个过滤器用1ml冰冷的水洗涤4次。
    1. 将完成测定所需的HAWP膜过滤器置于具有水以预湿润的培养皿中。根据需要去除过滤器,并在停止摄取测定之前将其置于过滤装置上。
    2. 重要的是避免将细胞悬浮液加载在过滤器边缘附近,因为如果过滤装置不能紧密地围绕过滤器,则可能导致细胞损失。
  7. 放射性测量


放射性底物的摄取以nmol底物/10 9个细胞对时间作图。对于每个摄取时间,进行三次重复测定,平均,并绘制平均值±标准偏差。为了确定特异性摄取,从总摄取中减去阴性对照值。放射性标记的Leu-Leu的摄取的代表图如下所示(图1)



  1. 上述实验测量底物进入细胞的积累或摄取。如果要进行运输的动力学测量,则必须了解所研究的底物的胞内命运。例如,如果肽在运输期间或在进入细胞质后立即快速水解,则通过上述方法测定动力学是无效的。还必须确定底物在运输前也不在细胞外代谢。如果发生细胞外水解,那么底物不适合摄取测量,因为放射性的摄取将测量氨基酸吸收。因此,在运输测量后,细胞外介质和细胞内内容物的提取和分析以确定底物的物理状态对于动力学运输研究是必要的,而如果仅评估"吸收"是研究的目标,则这种测定不是必要。
  2. 该实验可以用不同量的非代谢放射性底物重复,以通过Michaelis-Menten型分析确定摄取动力学。在通过在不同底物浓度下测量摄取对时间(图1)确定的积累的线性阶段中的时间测量吸收。对于每个摄取点,进行三次测定,平均,并绘制平均值±标准偏差。底物浓度(S)对吸收(V)的图将得到所研究的转运系统的表观K max和表观V max max。 V max表示由系统实现的最大速率,K max是摄取速率为最大值一半时的底物浓度。下面提供了运输动力学的代表性分析(图2)。

    图2.肽转运的动力学。在30℃下测定作为[3 H] Leu-Leu浓度(S)的函数的吸收(V)(总吸收)和0℃(阴性对照)。将值标准化以反映每10 9个酵母细胞的nmol摄取。以红色表示的比摄取通过从总数中减去阴性对照来确定。通过非线性回归拟合数据,以确定K sub(118±18nM)和预测的V max max(82±4nmol/10 细胞/min)的值


  1. 最小脯氨酸(MP)肉汤(1L)
    20克葡萄糖 不含(NH 4)2 SO 4和氨基酸的1.7g酵母氮源(YNB) 1g脯氨酸作为氮源 将葡萄糖溶于900ml水中并高压灭菌
    一旦冷却,加入100ml过滤灭菌的10×YNB-脯氨酸储备液(17g YNB和在100ml水中的10g脯氨酸)
  2. MP +肉汤
    100ml MP肉汤
    1.0ml尿嘧啶(2mg/ml,在双蒸水中,过滤灭菌) 1.0ml硫酸腺嘌呤(2mg/ml 0.1N HCl,过滤灭菌) 0.2ml组氨酸(10mg/ml,过滤灭菌) 0.3ml亮氨酸(10mg/ml,过滤灭菌) 0.2ml色氨酸(10mg/ml 0.1N HCl,过滤灭菌) 注意:添加上述氨基酸和核碱基以满足酿酒酵母W303的营养缺陷需要。可以修改酵母菌株或待测试的毒性化合物的特定生长条件。
  3. 20%葡萄糖(100ml) 每100ml含有20g葡萄糖 将葡萄糖溶于双蒸水中
  4. 2%葡萄糖 在水中稀释20%葡萄糖溶液1:10
  5. 2x吸收介质
    2×培养基由2%葡萄糖和20mM柠檬酸钠 - 磷酸钾(pH 5.5)组成 补充有放射性标记的肽[2μCi/ml(<14 C)Leu-Leu]和非放射性标记的肽(150μMLeu-Leu)


该方案改编自我们以前的研究(Cai等人,2007; Cai等人,2006)。这项工作是由国家综合医学科学研究院GM-22087和GM-46520的资助。


  1. Cai,H.,Kauffman,S.,Naider,F.和Becker,JM(2006)。  Cai,H.,Hauser,M.,Naider,F.和Becker,JM(2007)。 两种肽转运蛋白的差异调节和底物偏好:< em > Saccharomyces cerevisiae 。 Eukaryot Cell   6(10):1805-1813。
  • 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: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hauser, M., Cai, H., Naider, F. and Becker, J. M. (2016). Uptake Assay for Radiolabeled Peptides in Yeast. Bio-protocol 6(22): e2026. DOI: 10.21769/BioProtoc.2026.