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Jan 2018

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Analysis of Protein Stability by Synthesis Shutoff
通过合成关闭分析蛋白质稳定性   

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Abstract

In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are extracted via trichloroacetic acid (TCA) precipitation and subsequently separated via SDS-PAGE. Immunoblotting and antibody-decoration are performed to detect Fzo1 using HA-specific antibodies. We have adapted the method of blocking protein translation with cycloheximide to analyze the stability of high molecular weight proteins, including post-translational modifications and their impact on protein turnover.

Keywords: Shutoff (停止), Cycloheximide chase (环己酰亚胺追踪), Western blot (蛋白质印迹), SDS-PAGE (十二烷基硫酸钠聚丙烯酰胺凝胶电泳), Fzo1 (Fzo1)

Background

The stability of any protein of interest can be examined, after blocking translation, by following its fate over time. Unstable proteins undergo either proteasomal or vacuolar degradation. Thus, the physiological relevance of protein turnover can be deciphered by blocking the proteasome, the vacuolar proteases, or by using stable mutant variants thereof. To bypass the need for antibodies binding specifically to it, tagged versions of the protein of interest can be constructed and expressed.


Here, we provide a detailed protocol to assess protein stability and its dependence on the proteasome. A low-cost and reliable western blot-based assay is presented, benefiting from the turnover properties of the protein Fzo1. The yeast mitofusin Fzo1 is a known target of post-translational ubiquitylation (Schuster et al., 2018), which is degraded under normal physiological conditions (Anton et al., 2013; Anton et al., 2011; Anton et al., 2019; Schuster et al., 2020; Simoes et al., 2018). Residues in Fzo1 and enzymes required for its turnover have been identified, whereby the use of respective mutations preventing this degradation can be used as a turnover control. In addition, increased instability, which depends on the proteasome, can happen during stress conditions or upon genetic alterations. Thus, as described below and exemplified in the representative figure, Fzo1 represents a good case-study example for protein stability analysis. Our protocol provides a detailed step-by-step description to analyze protein stability with a non-toxic preparation procedure and lower amounts of cycloheximide when compared to the commonly used protocol fromBuchanan et al. (2016). Cycloheximide (CHX) is widely known to block translational elongation by interfering with deacetylated tRNA and binding the ribosome on the 60S subunit (Klinge et al., 2011; Schneider-Poetsch et al., 2010). Furthermore, our protein extraction protocol using trichloroacetic acid (TCA) can be used for a western blot-based analysis of both small and high molecular weight proteins.

Materials and Reagents

  1. Filter papers (Macherey-Nagel, catalog number: 742113)

  2. Nitrocellulose membrane (Amersham, catalog number: 10600001)

  3. Centrifugation tubes (15 ml, 1.5 ml) (VWR, catalogue number: 352096 [15 ml], Sarstedt, catalog number: 72.690.001 [1.5 ml])

  4. X-ray films (Fujifilm, catalog number: 47410 19289)

  5. Yeast Saccharomyces cerevisiae BY4741 isogenic to S288c (Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (Brachmann et al., 1998)

  6. D(+)-Glucose monohydrate (Roth, catalog number: 6780.2)

  7. Cycloheximide InSolution 100 mg/ml in DMSO (Sigma, catalog number: C4859)

  8. MG132 InSolution 10 mM in DMSO (Calbiochem, catalog number: 474791)

  9. NaOH (Merck, catalog number: 1.06498.5000)

  10. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250-100ML)

  11. Trichloroacetic acid (TCA) (Roth, catalog number: 3744.1)

  12. SDS (Roth, catalog number: CN30.3)

  13. Tris (Roth, catalog number: 5429.5)

  14. NaCl (Roth, catalog number: 3957.2)

  15. 37% HCl (VWR, catalog number: 20252.335)

  16. Glycine (PanReac AppliChem, catalog number: 141340.0914)

  17. DTT (MP, catalog number: 100597)

  18. Bromophenol Blue sodium salt (USB, catalog number: US12370)

  19. Glycerol (Serva, catalog number: 23176.01)

  20. Color Prestained Protein Standard, Broad Range 11-245 kDa (New England Biolabs, catalog number: P7719)

  21. Rotiphorese Gel 30 Acrylamide/Bis-acrylamide stock solution (Roth, catalog number: 3029.1)

  22. Ammonium Peroxodisulfate (APS) (Merck, catalog number: 1.01201.0500)

  23. Tetramethylethylenediamine (TEMED) (Sigma, catalog number: T9281-25ML)

  24. Methanol (Roth, catalog number: 8388.5)

  25. 2-Propanol (VWR, catalog number: 20842.330)

  26. Ponceau S (Serva, catalog number: 33429.02)

  27. Acetic acid (VWR, catalog number: 20104.334)

  28. Nonfat dried milk powder (Roth, catalog number: T145.3)

  29. HA primary antibody (clone 3F10) (Roche, catalog number: 11867423001)

  30. Sodium Azide (NaN3) (Merck, catalog number: 1.06688.0100)

  31. Rat secondary antibody (Invitrogen, catalog number: 31470)

  32. GE Healthcare AmershamTM ECLTM Prime (Amersham, catalog number: RPN2232)

  33. TBS Buffer, pH 7.4 (see Recipes)

  34. HU sample buffer (see Recipes)

  35. Ponceau S solution (see Recipes)

  36. Tris/Glycine buffer (see Recipes)

  37. Transfer buffer (see Recipes)

  38. Minimal yeast media which allows auxotrophic selection of yeast cells (see Recipes)

  39. 1 M DTT (see Recipes)

  40. Rödel mix (see Recipes)

Equipment

  1. Shaking incubator for yeast cultures (Infors-HT, Multitron Standard)

  2. Bench-top centrifuge (VWR, model: Mega star 1.6)

  3. Bench-top cooling centrifuge (Eppendorf, model: 5145R)

  4. Vortex (Scientific Industries, Inc., model: Vortex-Genie 2)

  5. Photometer (Hitachi, model: Double Beam Spectrophotometer U-2900)

  6. Thermomixer (Eppendorf, model: Thermomixer comfort 5355)

  7. Glass syringe (Hamilton, e.g., catalog number: 80565)

    Note: Instead of a Hamilton syringe, capillary pipette tips (VWR, catalog number: 53509-015) can be used.

  8. SDS-PAGE equipment (HoeferTM, model: SE 600 Series)

  9. Western blot equipment (Peqlab, PerfectBlueTM, model: “semi-dry”-Blotter, SedecTM)

  10. Tumbling table (Biometra, model: WT12)

  11. Autoradiography cassette (Amersham, model: RPN11642)

  12. Developer machine (AGFA, model: CURIX 60)

Procedure

  1. Culture conditions

    1. Preculture (Day 1)

      Inoculate a small number of cells [starting optical density (OD)600 0.05 = 750,000 cells/ml] in 5 ml of selective minimal media supplemented with glucose (2%, w/v) and grow overnight to the exponential growth phase at 30°C, shaking at 160 rpm.

      Note: 30°C is the optimal growth condition for wild-type yeast strains. For the analysis of essential proteins, and therefore genetically manipulated temperature-sensitive (ts) yeast strains, the growth temperature must be adapted accordingly. Furthermore, some proteins might be degraded only under certain conditions, for example, upon heat shock, which should therefore be tested for each case.

    2. Main culture (Day 2)

      Inoculate 50 ml of selective media supplemented with glucose (2%, w/v) with a starting OD600 = 0.3 in the morning, by diluting the yeast cells from the overnight culture. Let the cells divide at least 4-6 h to the exponential growth phase, up to a maximum OD600 = 2.


  2. Protein synthesis shutoff by CHX treatment

    1. Harvest cells an OD600 = 12 in total: measure the OD of the main culture and calculate the necessary volume (V) in milliliters by using V = 12 /measured OD600 of the main culture. Use 15 ml conical tubes for centrifugation (5 min, 4,100 × g, room temperature), keep 4 ml of growth media inside the tube, and discard the rest of the supernatant.


      Example:

      1. Measured OD600 of main culture = 1.3

      2. Needed (V): 12 OD600 in ml

        Calculate: V = 12 OD600/1.3 OD600 = 9.23 ml

      3. 9.23 ml will be transferred to a 15ml conical tube and cells harvested by centrifugation


    2. Resuspend the harvested cells in 4 ml of media.

    3. This step is optional, depending on the need for blocking the proteasome before translation inhibition. To inhibit the proteasome, 0.1 mM MG132 is added to the media, and cells are incubated for 1 h at 30°C, shaking at 160 rpm.

      Note: If proteasomal inhibition is performed by MG132 treatment, cells have to be previously grown for at least 3 h in media containing proline and 0.003% SDS (Liu et al., 2007), to ensure that the MG132 is not exported from the cells immediately via several ABC (ATP binding cassette) transporters (Liu et al., 2007). Alternatively, cells lacking the ABC transporter Pdr5 and/or Snq2 can be used to perform proteasomal inhibition without using special culturing conditions. Importantly: Cells deleted for PDR5/SNQ2 are treated with only 0.05 mM MG132 due to increased drug permeability (Collins et al., 2010).

    4. Add CHX to the cells to a final concentration of 100 µM and invert tubes gently up to five times.

    5. Immediately transfer 1 ml of cell suspension containing 3 OD600 to a new reaction tube and harvest cells by centrifugation (5min, 16,100 × g, 4°C). Aspirate the supernatant and freeze the cell pellet at -20°C (time point (t) = 0).

    6. Shake cells again for 1 h and afterwards repeat Step B5 (t = 1).

    7. After an additional incubation of 2 h at 30°C, shaking at 160 rpm, 1 ml of media containing 3 OD600 will be harvested again (5 min, 16,100 × g, 4°C) (t = 3).

      Note: The time points of the chase should be adjusted to the protein used in the study, e.g., a very unstable protein should be analyzed in a time frame of several minutes to 1 h, while stable proteins should be analyzed over several hours.


  3. Sample preparation

    Note: All following steps are performed on ice.

    1. Cell pellets are resuspended in 1ml of sterile distilled water and 150 µl of Rödels mix, to break up the yeast cell wall and denature proteins by vortexing and incubating for 15 min on ice.

    2. To precipitate the proteins, add 150 µl of 55% TCA to each sample. Invert the tubes and incubate for 10 min on ice.

    3. Centrifuge for 10 min, 16,100 × g at 4°C.

    4. Carefully remove the supernatant.

    5. Centrifuge again 5 min, 16,100 × g at 4°C.

    6. Remove the whole supernatant and resuspend the pellet in 50 µl of HU sample buffer (see Recipes) by shaking tubes for 20 min, at 45°C and with 1,400 rpm on a thermomixer.


  4. SDS-PAGE and Immunoblotting

    Proteins are separated using SDS-PAGE, as previously described (Schuster et al., 2018).

    1. Gel equipment is assembled according to the manufacturer’s instructions.

    2. Gel mixtures for stacking and separation gels are prepared as described in Table 1 but without APS and TEMED.

      Note: The gel used to analyze the protein via SDS-PAGE depends on the size of the protein of interest. The smaller the size of the protein, the higher the percentage of the acrylamide and vice versa (Sambrook and Russell, 2006).


      Table 1. Composition of one 12% SDS gel

      12% Separation gel (ml) Stacking gel (ml)
      Acrylamide1 4 0.5
      1.875 M Tris pH 8.8 2 -
      0.6M Tris pH 6.8 - 0.6
      MilliQ water 3.8 1.85
      10% SDS 0.05 0.02
      20% APS 0.05 0.015
      TEMED 0.01 0.005

      1 30% Acrylamide with 0.8% Bisacrylamide (37.5:1)


    3. Add APS and TEMED to the separation gel mix, mix rapidly and thoroughly, and pour 9.5 ml of the mixture between two glass plates. Add 1 ml of 2-propanol on top of the gel mix to remove bubbles and smoothen the surface. Let the gel polymerize for at least 15 min.

    4. Remove 2-propanol and wash 3× carefully with distilled water. Carefully remove excess water with a filter paper.

    5. Put in the combs to the space between both glass plates before pouring the upper gel. Add APS and TEMED to the stacking gel mix. Mix well and pour the stacking gel mix on top of the separation gel until the space between both glass plates is full.

    6. After polymerization of the upper stacking gel mixture (for at least 15 minutes), remove the combs and fill the wells with Tris/Glycine buffer.

    7. Load a maximum of 15 µl per sample and include a pre-stained protein standard marker (e.g., NEB, P7719) in each gel.

    8. Fill the lower chamber of the electrophoresis tank (anode) with Tris/Glycine buffer and insert gels into the system according to the manufacturer’s instructions.

    9. Fill the upper cathode chamber with freshly prepared Tris/Glycine buffer. Follow the manufacturer’s instructions for information on volumes of buffers required.

    10. Run gels at 200 V and 400 mA until the protein loading front (visible due to the bromophenol blue sodium salt in the HU-buffer, used as a tracking dye to monitor the progress of molecules moving through the polyacrylamide gel) reaches the end of the gel. To estimate the size of proteins, refer to the pre-stained protein standard on the gel. Ensure that the protein of interest stays inside the gel and does not run out.

    11. Remove the gels from the running systems, disassemble the glass plate sandwich, and remove the stacking gel with a plastic wedge.

    12. Transfer the acrylamide gels onto nitrocellulose membranes via semi-dry western blotting, as previously described in Schuster et al. (2018) .

    13. Incubate membranes in Ponceau S (PoS) solution to stain all proteins transferred to the membrane. This staining is used to ascertain sufficient protein transfer and serves as a loading control. After imaging the membrane, destain it in 20 ml of TBS for at least 15 minutes.

    14. Perform antibody detection. In the case exemplified in this protocol, HA-specific antibodies were used. Dilute the antibodies 1:1,000 in 5% milk in TBS. Incubate the membranes in the primary antibody overnight, at 4°C. The next day, remove the primary antibody, wash the membranes for 15 min with TBS, followed by 2 h incubation at room temperature with the secondary α-rat antibody diluted 1:5,000 in 5% milk in TBS. After removing the secondary antibody, wash the membrane with TBS three times for 10 min. The secondary antibody is coupled to the enzyme horseradish peroxidase (HRP), used to create an emission of light in a hydrogen peroxide-catalyzed oxidation of luminol. Western blots are developed by using light sensitive X-ray films and an enhanced chemiluminescence solution (Cytiva AmershamTM ECLTM Prime), containing hydrogen peroxide and luminol, as previously described in the protocol from Schuster et al. (2018) .

      Note: Degradation of an unstable protein (e.g., Ubc6) during CHX treatment or its stabilization upon MG132 treatment can be used as a positive control for translational and/or proteasomal inhibition, respectively.


      In Figure 1, a representative image is shown.



    Figure 1. Stability of Fzo1 after translational shut down, with or without proteasomal inhibition.

    (A) Cells deleted for FZO1 and expressing HA-tagged Fzo1 were compared to cells expressing a mutant version of Fzo1 called mutant-1, which stabilizes the protein. (B) Cells lacking PDR5 and stably expressing genomically tagged versions of either HA-tagged wt Fzo1 or an unstable mutant version, called mutant-2, were analyzed. Cells were treated with 100 µM CHX for 1 or 3 h. When indicated, cells were incubated for 1 h with 0.05 mM MG132 prior to CHX treatment. (A+B) Protein levels were analyzed in total cellular extracts by SDS-PAGE and immunoblotting. Fzo1 was detected by using HA-specific antibodies. The unstable ER-protein Ubc6, observed in this analysis by using Ubc6-specific antibodies, was used as a control for proteasomal inhibition with MG132 and for translational inhibition with CHX (representative image). Ponceau S (PoS) staining was used as a loading control.

Recipes

  1. TBS Buffer, pH 7.4

    50 mM Tris

    150 mM NaCl

    Adjust pH to 7.4 using 37% HCl

  2. HU sample buffer

    8 M Urea

    5% SDS

    200 mM Tris pH 6.8

    0.2 g/L Bromophenol blue sodium salt

    100 mM DTT added freshly before use

  3. Ponceau S solution

    0.1% Ponceau S

    5% Acetic acid

  4. Tris/Glycine buffer

    25 mM Tris

    200mM Glycine

    3.5mM SDS

  5. Transfer buffer

    200 mM Glycine

    25 mM Tris

    20% (v/v) Methanol

    0.02% SDS

    Adjust pH to 8.5 using 37% HCl

  6. Minimal yeast media, which allows auxotrophic selection of yeast cells (Kaiser et al., 1994)

    1.5 g Drop out mix (containing all amino acids except Adenine, Uracil, Leucine, Lysine, Histidine, and Tryptophane)

    1.7 g Yeast Nitrogen Base

    5 g Ammonium sulfate

    Components listed above are dissolved in 1 l of distilled water, autoclaved and supplemented with 2% glucose and a CSM (complete supplement mixture) amino acid mix (containing Ade, Ura, Lys, His, Trp, Leu).

  7. 1 M DTT

    1.54 g DTT in 10 ml of in sterile distilled water

  8. Rödel mix

    2 M NaOH

    7.5% β-mercaptoethanol

Acknowledgments

Funding: Deutsche Forschungsgemeinschaft (DFG; CRC 1218 TP A03; EXC 2030 – 390661388; to ME-H); Center for Molecular Medicine Cologne (CMMC; CAP14, to ME-H and A02 to ME-H and M. Odenthal); Boehringer Ingelheim foundation (BIS; Exploration grant; to ME-H). This protocol was used in Simões et al. (2018; Doi: 10.7554/eLife.30015).

Competing interests

The authors declare they have no conflict of interest or competing interests.

References

  1. Anton, F., Dittmar, G., Langer, T. and Escobar-Henriques, M. (2013). Two deubiquitylases act on mitofusin and regulate mitochondrial fusion along independent pathways. Mol Cell 49(3): 487-498.
  2. Anton, F., Fres, J. M., Schauss, A., Pinson, B., Praefcke, G. J., Langer, T. and Escobar-Henriques, M. (2011). Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion. J Cell Sci (7): 1126-1135.
  3. Anton, V., Buntenbroich, I., Schuster, R., Babatz, F., Simoes, T., Altin, S., Calabrese, G., Riemer, J., Schauss, A. and Escobar-Henriques, M. (2019). Plasticity in salt bridge allows fusion-competent ubiquitylation of mitofusins and Cdc48 recognition. Life Sci Alliance 2(6): e201900491.
  4. Buchanan, B. W., Lloyd, M. E., Engle, S. M. and Rubenstein, E. M. (2016). Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae. J Vis Exp (110): 53975.
  5. Collins, G. A., Gomez, T. A., Deshaies, R. J. and Tansey, W. P. (2010). Combined chemical and genetic approach to inhibit proteolysis by the proteasome. Yeast 27(11): 965-974.
  6. Kaiser, C., Michaelis, S. and Mitchell, A. (1994). Methods in yeast genetics: a Cold Spring Harbor laboratory course manual. Plainview: Cold Spring Harbor Laboratory.
  7. Klinge, S., Voigts-Hoffmann, F., Leibundgut, M., Arpagaus, S. and Ban, N. (2011). Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor. Science 334(6058): 941-948.
  8. Liu, C., Apodaca, J., Davis, L. E. and Rao, H. (2007). Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells. Biotechniques 42(2): 158, 160, 162.
  9. Sambrook, J. and Russell, D. W. (2006). SDS-Polyacrylamide Gel Electrophoresis of Proteins. CSH Protoc 2006(4): pdb.prot4540.
  10. Schneider-Poetsch, T., Ju, J., Eyler, D. E., Dang, Y., Bhat, S., Merrick, W. C., Green, R., Shen, B. and Liu, J. O. (2010). Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6(3): 209-217.
  11. Schuster, R., Anton, V., Simoes, T., Altin, S., den Brave, F., Hermanns, T., Hospenthal, M., Komander, D., Dittmar, G., Dohmen, R. J. and Escobar-Henriques, M. (2020). Dual role of a GTPase conformational switch for membrane fusion by mitofusin ubiquitylation. Life Sci Alliance 3(1): e201900476.
  12. Schuster, R., Simões, T., den Brave, F. and Escobar-Henriques, M. (2018). Separation and Visualization of Low Abundant Ubiquitylated Forms. Bio-protocol 8(22): e3081.
  13. Simões, T., Schuster, R., den Brave, F. and Escobar-Henriques, M. (2018). Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion. Elife 7: e30015.

简介

[摘要]在这个协议中,我们描述了蛋白质稳定性随时间的分析,使用合成关闭。例如,我们在酿酒酵母中表达 HA 标记的酵母丝裂融合蛋白 Fzo1,并通过放线菌酮 (CHX) 抑制翻译。在添加 CHX 之前,作为可选步骤,使用 MG132 进行蛋白酶体抑制。通过三氯乙酸 (TCA) 沉淀提取蛋白质,然后通过 SDS-PAGE 分离。使用 HA 特异性抗体进行免疫印迹和抗体修饰以检测 Fzo1。我们采用了用放线菌酮阻断蛋白质翻译的方法来分析高分子量蛋白质的稳定性,包括翻译后修饰及其对蛋白质周转的影响。

[背景] 在阻断翻译后,可以通过跟踪其随时间的变化来检查任何感兴趣的蛋白质的稳定性。不稳定的蛋白质会经历蛋白酶体或液泡降解。因此,蛋白质周转的生理相关性可以通过阻断蛋白酶体、液泡蛋白酶或通过使用其稳定的突变变体来破译。为了绕过抗体与其特异性结合的需要,可以构建和表达目标蛋白质的标记版本。
在这里,我们提供了一个详细的协议来评估蛋白质稳定性及其对蛋白酶体的依赖。介绍了一种低成本且可靠的基于蛋白质印迹的测定,受益于蛋白质 Fzo1 的周转特性。酵母丝裂蛋白 Fzo1 是翻译后泛素化的已知靶标(Schuster等,2018),在正常生理条件下会降解(Anton等,2013;Anton等,2011;Anton等,2019) ;Schuster等人,2020 年;Simoes等人,2018 年)。Fzo1 中的残留物和其周转所需的酶已被确定,从而可以使用防止这种降解的相应突变作为周转控制。此外,取决于蛋白酶体的不稳定性增加可能发生在压力条件下或基因改变时。因此,如下文所述并在代表性图中举例说明,Fzo1 代表了蛋白质稳定性分析的一个很好的案例研究示例。我们的协议提供了详细的分步描述,与布坎南等人的常用协议相比,通过无毒制备程序和较少量的放线菌酮来分析蛋白质稳定性。(2016)。众所周知,放线菌酮 (CHX) 通过干扰去乙酰化的 tRNA 并结合 60S 亚基上的核糖体来阻止翻译延伸(Klinge等人,2011 年;Schneider-Poetsch等人,2010 年)。此外,我们使用三氯乙酸 (TCA) 的蛋白质提取协议可用于对小分子量和高分子量蛋白质进行基于蛋白质印迹的分析。

关键字:停止, 环己酰亚胺追踪, 蛋白质印迹, 十二烷基硫酸钠聚丙烯酰胺凝胶电泳, Fzo1

材料和试剂

 

1.     滤纸(Macherey-Nagel,目录号:742113

2.     硝酸纤维素膜(Amersham,目录号:10600001

3.     离心管(15 ml1.5 ml)(VWR,目录号:352096 [15 ml]Sarstedt,目录号:72.690.001 [1.5 ml]

4.     X 射线胶片(Fujifilm,目录号:47410 19289

5.     酵母Saccharomyces cerevisiae BY4741 S288c 同基因 (Mata his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ ) (Brachmann et al. , 1998)

6.     D+-葡萄糖一水合物(Roth,目录号:6780.2

7.     放线菌酮溶液 100 mg/ml DMSOSigma,目录号:C4859

8.     MG132 InSolution 10 mM DMSO 中(Calbiochem,目录号:474791

9.     NaOH(默克,目录号:1.06498.5000

10.  β-巯基乙醇(Sigma-Aldrich,目录号:M6250-100ML

11.  三氯乙酸(TCA)(Roth,目录号:3744.1

12.  SDSRoth,目录号:CN30.3

13.  TrisRoth,目录号:5429.5

14.  NaClRoth,目录号:3957.2

15.  37% HClVWR,目录号:20252.335

16.  甘氨酸(PanReac AppliChem,目录号:141340.0914

17.  DTTMP,目录号:100597

18.  溴酚蓝钠盐(USB,目录号:US12370

19.  甘油(Serva,目录号:23176.01

20.  彩色预染蛋白质标准品,宽范围 11-245 kDaNew England Biolabs,目录号:P7719

21.  Rotiphorese Gel 30 丙烯酰胺/双丙烯酰胺原液(Roth,目录号:3029.1

22.  过二硫酸铵(APS)(默克,目录号:1.01201.0500

23.  四甲基乙二胺(TEMED)(Sigma,目录号:T9281-25ML

24.  甲醇(Roth,目录号:8388.5

25.  2-丙醇(VWR,目录号:20842.330

26.  Ponceau SServa,目录号:33429.02

27.  乙酸(VWR,目录号:20104.334

28.  脱脂奶粉(Roth,目录号:T145.3

29.  HA 一抗(克隆 3F10)(Roche,目录号:11867423001

30.  叠氮化钠(NaN )(默克,目录号:1.06688.0100

31.  大鼠二抗(Invitrogen,目录号:31470

32.  GE Healthcare Amersham TM ECL TM PrimeAmersham,目录号:RPN2232

33.  TBS 缓冲液,pH 7.4(见配方)

34.  HU 样品缓冲液(见配方)

35.  Ponceau S 溶液(见配方)

36.  Tris/甘氨酸缓冲液(见配方)

37.  转移缓冲液(见配方)

38.  允许对酵母细胞进行营养缺陷选择的最小酵母培养基(参见食谱)

39.  1 M DTT(见食谱)

40.  Rödel 混合(见食谱)

 

设备

 

1.     酵母培养物的振动培养箱(Infors-HTMultitron Standard

2.     台式离心机(VWR,型号:Mega star 1.6

3.     台式冷却离心机(Eppendorf,型号:5145R

4.     VortexScientific Industries, Inc.,型号:Vortex-Genie 2

5.     光度计(日立,型号:双光束分光光度计 U-2900

6.     ThermomixerEppendorf,型号:Thermomixer Comfort 5355

7.     玻璃注射器(Hamilton例如,目录号:80565

注意:可以使用毛细管吸头(VWR,目录号:53509-015)代替 Hamilton 注射器。

8.     SDS-PAGE 设备(Hoefer TM ,型号:SE 600 系列)

9.     蛋白质印迹设备(PeqlabPerfectBlue TM ,型号:半干”-BlotterSedec TM 

10.  翻滚桌(Biometra,型号:WT12

11.  放射自显影盒(Amersham,型号:RPN11642

12.  显影机(AGFA,型号:CURIX 60

 

程序

 

A.    培养条件

1.     预培养(第 1 天)

接种少量细胞 [起始光密度 (OD) 600 0.05 = 750,000 个细胞/毫升] 5 毫升补充有葡萄糖 (2%, w/v) 的选择性基本培养基中,并在 30° 下过夜生长至指数生长期C160/分振摇。

注意:30°C 是野生型酵母菌株的最佳生长条件。对于必需蛋白质的分析,因此对基因操作的温度敏感 (ts) 酵母菌株进行分析,必须相应地调整生长温度。此外,某些蛋白质可能仅在某些条件下降解,例如在热休克时,因此应对每种情况进行测试。

2.     主要文化(第 2 天)

通过稀释过夜培养物中的酵母细胞,在早上接种 50 ml 补充有葡萄糖(2%w/v)的选择性培养基,起始 OD 600 = 0.3。让细胞分裂至少 4-6 小时到指数生长期,最大 OD 600 = 2

 

B.    CHX处理关闭蛋白质合成

1.     收获细胞总OD 600 = 12:测量主要培养物的 OD 并使用 V = 12 / 测量的主要培养物的OD 600计算所需的体积 (V),以毫升为单位。使用 15 ml 锥形管进行离心(5 分钟,4,100 × g ,室温),在管内保留 4 ml 生长培养基,并丢弃剩余的上清液。

 

例子:                            

·  主要培养物的实测 OD 600 = 1.3

·  需要 (V)12 OD 600毫升

计算:V = 12 OD 600 /1.3 OD 600 = 9.23 ml

·  9.23 ml 将转移到 15 ml 锥形管中,并通过离心收集细胞

 

2.     4 ml 培养基中重悬收获的细胞。

3.     该步骤是可选的,取决于在翻译抑制之前阻断蛋白酶体的需要。为了抑制蛋白酶体,将 0.1 mM MG132 添加到培养基中,并将细胞在 30°C 下孵育 1 小时,以 160 rpm 振荡。

注意:如果蛋白酶体抑制是通过 MG132 处理进行的,细胞必须预先在含有脯氨酸和 0.003% SDS 的培养基中生长至少 3 小时(Liu 等,2007),以确保 MG132 不会从细胞中输出立即通过几个 ABCATP 结合盒)转运蛋白(Liu 等,2007)。或者,可以使用缺乏 ABC 转运蛋白 Pdr5 / Snq2 的细胞进行蛋白酶体抑制,而无需使用特殊的培养条件。重要的是:由于药物渗透性增加,PDR5/SNQ2 缺失的细胞仅用 0.05 mM MG132 处理(Collins 等,2010)。

4.     CHX 添加到细胞中,最终浓度为 100 µM,并轻轻地将管反转五次。

5.     立即将 1 ml 含有 3 OD 600的细胞悬液转移到新的反应管中,并通过离心(5 分钟,16,100 × g 4°C)收获细胞。吸出上清液并在 -20°C 下冷冻细胞沉淀(时间点 (t) = 0)。

6.     再次摇动细胞 1 小时,然后重复步骤 B5t = 1)。

7.     30°C 下再孵育 2 小时后,以 160 rpm 摇动,将再次收获1 ml 含有 3 OD 600的培养基(5 分钟,16,100 × g 4°C)(t = 3)。

注意:追逐的时间点应根据研究中使用的蛋白质进行调整,例如,应在几分钟到 1 小时的时间范围内分析非常不稳定的蛋白质,而应在几个小时内分析稳定的蛋白质。

 

 

C.    样品制备

注意:以下所有步骤均在冰上进行。

1.     将细胞沉淀重悬于 1ml 无菌蒸馏水和 150 μl Rödels 混合物中,通过涡旋和冰上孵育 15 分钟来破坏酵母细胞壁并使蛋白质变性。

2.     要沉淀蛋白质,请向每个样品中加入 150 µl 55% TCA。倒置试管并在冰上孵育 10 分钟。

3.     4°C 下以16,100 × g离心 10 分钟。

4.     小心去除上清液。

5.     4°C 下再次离心 5 分钟,16,100 × g 

6.     45°C 1,400 rpm 的热混合器上摇动管 20 分钟,去除整个上清液并在 50 µl HU 样品缓冲液(参见配方)中重悬沉淀。

 

D.    SDS-PAGE 和免疫印迹

如前所述(Schuster2018),使用 SDS-PAGE 分离蛋白质。

1.     凝胶设备根据制造商的说明组装。

2.     用于堆积和分离凝胶的凝胶混合物按表 1 所述制备,但不含 APS TEMED

注意:用于通过 SDS-PAGE 分析蛋白质的凝胶取决于目标蛋白质的大小。蛋白质的尺寸越小,丙烯酰胺的百分比越高,反之亦然(Sambrook Russell2006)。

 

1.一块 12% SDS 凝胶的组成


12% 分离胶 (ml)

堆积凝胶 (ml)

丙烯酰胺1

4

0.5

1.875 M Tris pH 8.8

2

——

0.6M Tris pH 6.8

——

0.6

MilliQ

3.8

1.85

10% 安全数据表

0.05

0.02

20% APS

0.05

0.015

泰德

0.01

0.005

30% 丙烯酰胺和 0.8% 双丙烯酰胺 (37.5:1)

 

3.     APS TEMED 添加到分离凝胶混合物中,快速彻底混合,然后将 9.5 ml 混合物倒入两个玻璃板之间。在凝胶混合物上加入 1 ml 2-丙醇以去除气泡并使表面光滑。让凝胶聚合至少 15 分钟。

4.     除去2-丙醇和洗涤3 ×小心地用蒸馏水。用滤纸小心地除去多余的水。

5.     在倒入上层凝胶之前,将梳子放入两块玻璃板之间的空间。将 APS TEMED 添加到浓缩凝胶混合物中。充分混合并将浓缩凝胶混合物倒在分离凝胶的顶部,直到两块玻璃板之间的空间已满。

6.     上层浓缩凝胶混合物聚合后(至少 15 分钟),取出梳子并用 Tris/甘氨酸缓冲液填充孔。

7.     每个样品最多上样 15 µl,并在每个凝胶中包含一个预染色的蛋白质标准标记物(例如NEBP7719)。

8.     Tris/甘氨酸缓冲液填充电泳槽(阳极)的下室,并根据制造商的说明将凝胶插入系统。

9.     用新鲜制备的 Tris/甘氨酸缓冲液填充上阴极室。请按照制造商的说明获取有关所需缓冲液体积的信息。

10.  200 V 400 mA 下运行凝胶,直到蛋白质加载前沿(由于 HU 缓冲液中的溴酚蓝钠盐可见,用作跟踪染料以监测分子在聚丙烯酰胺凝胶中移动的进程)到达末端凝胶。要估计蛋白质的大小,请参阅凝胶上的预染色蛋白质标准。确保感兴趣的蛋白质留在凝胶内并且不会耗尽。

11.  从运行系统中取出凝胶,拆卸玻璃板三明治,并用塑料楔子取出堆积凝胶。

12.  通过半干西方印迹将丙烯酰胺凝胶转移到硝酸纤维素膜上,如先前在 Schuster等人中所述(2018)

13.  Ponceau S (PoS) 溶液中孵育膜以染色转移到膜上的所有蛋白质。这种染色用于确定足够的蛋白质转移并用作加载控制。膜成像后,将其在 20 毫升 TBS 中脱色至少 15 分钟。

14.  进行抗体检测。在本协议中举例说明的情况下,使用了 HA 特异性抗体。在 TBS 中的 5% 牛奶中以 1:1,000 的比例稀释抗体。在 4°C 下将一抗中的膜孵育过夜。第二天,去除一抗,用 TBS 清洗膜 15 分钟,然后在室温下用二抗在 TBS 中的 5% 牛奶中稀释 1:5,000 孵育 2 小时。去除二抗后,用 TBS 洗膜 3 次,每次 10 分钟。二抗与辣根过氧化物酶 (HRP) 偶联,用于在过氧化氢催化的鲁米诺氧化中产生光发射。蛋白质印迹是通过使用光敏 X 射线胶片和增强的化学发光溶液 (Cytiva Amersham TM ECL TM Prime) 开发的,其中包含过氧化氢和鲁米诺,如先前 Schuster等人的协议中所述(2018)

注意:CHX 处理期间不稳定蛋白质(例如 Ubc6)的降解或其在 MG132 处理后的稳定化可分别用作翻译和/或蛋白酶体抑制的阳性对照。


 在图 1 中,显示了一个代表性图像。

 

 

1. 翻译关闭后 Fzo1 的稳定性,有或没有蛋白酶体抑制。

(A) FZO1删除并表达 HA 标记的 Fzo1 的细胞与表达 Fzo1 突变版本的细胞进行比较,称为突变体-1 ,稳定蛋白质。(B) 分析了缺乏PDR5并稳定表达基因组标记版本的 HA 标记的 wt Fzo1 或称为突变体-2的不稳定突变体版本的细胞。细胞用 100 µM CHX 处理 1 3 小时。当指示时,在 CHX 处理之前,将细胞与 0.05 mM MG132 孵育 1 小时。(A+B) 通过 SDS-PAGE 和免疫印迹分析总细胞提取物中的蛋白质水平。Fzo1 是通过使用 HA 特异性抗体检测到的。在该分析中通过使用 Ubc6 特异性抗体观察到的不稳定 ER 蛋白 Ubc6 被用作 MG132 蛋白酶体抑制和 CHX 翻译抑制的对照(代表性图像)。丽春红 S (PoS) 染色用作上样对照。

 

食谱

 

1.     TBS 缓冲液,pH 7.4

50 mM Tris

150 毫米氯化钠

使用 37% HCl pH 值调节至 7.4

2.     HU 样品缓冲液

8M尿素

5% 安全数据表

200 mM Tris pH 6.8

0.2 g/L 溴酚蓝钠盐

使用前新鲜添加 100 mM DTT

3.     丽春红 S 溶液

0.1% 春红 S

5% 醋酸

4.     Tris/甘氨酸缓冲液

25 mM Tris

200mM 甘氨酸

3.5mM SDS

5.     传输缓冲区

200 mM 甘氨酸

25 mM Tris

20% (v/v) 甲醇

0.02% SDS

使用 37% HCl pH 值调节至 8.5

6.     最低限度的酵母培养基,允许对酵母细胞进行营养缺陷型选择(Kaiser1994

1.5 g Dropout mix(包含除腺嘌呤、尿嘧啶、亮氨酸、赖氨酸、组氨酸和色氨酸以外的所有氨基酸)

1.7 克酵母氮基

5克硫酸铵

上面列出的成分溶解在 1 升蒸馏水中,高压灭菌并补充 2% 葡萄糖和 CSM(完全补充混合物)氨基酸混合物(包含 AdeUraLysHisTrpLeu)。

7.     1 M 数字电视

1.54 DTT 溶于 10 毫升无菌蒸馏水中

8.     罗德尔混合

2M氢氧化钠

7.5% β-巯基乙醇

 

致谢

 

资助:Deutsche Forschungsgemeinschaft (DFG; CRC 1218 TP A03; EXC 2030 – 390661388; to ME-H); 科隆分子医学中心(CMMCCAP14,到 ME-HA02 ME-H M. Odenthal);勃林格殷格翰基金会(BIS;勘探补助金;授予 ME-H)。Simões等人使用了该协议。(2018 年;Doi10.7554/eLife.30015)。

 

利益争夺

 

作者声明他们没有利益冲突或竞争利益。


参考

 

1.     Anton, F.Dittmar, G.Langer, T. Escobar-Henriques, M.2013 年)。两种去泛素酶作用于丝裂融合蛋白并沿独立途径调节线粒体融合。摩尔细胞493):487-498

2.     Anton, F., Fres, JM, Schauss, A., Pinson, B., Praefcke, GJ, Langer, T. Escobar-Henriques, M. (2011)Ugo1 Mdm30 Fzo1 导的线粒体外膜融合过程中依次起作用。J Cell Sci 124(第 7 篇):1126-1135              

3.     Anton, V., Buntenbroich, I., Schuster, R., Babatz, F., Simoes, T., Altin, S., Calabrese, G., Riemer, J., Schauss, A. Escobar-Henriques, M . (2019)盐桥中的可塑性允许有丝分裂蛋白和 Cdc48 识别的融合能力泛素化。生命科学联盟2(6)e201900491

4.     Buchanan, BW, Lloyd, ME, Engle, SM Rubenstein, EM (2016)酿酒酵母中蛋白质降解的放线菌酮追踪分析。J Vis Exp (110)53975

5.     Collins, GA, Gomez, TA, Deshaies, RJ Tansey, WP (2010)结合化学和遗传方法来抑制蛋白酶体的蛋白水解。酵母27(11)965-974              

6.     Kaiser, C.Michaelis, S. Mitchell, A. (1994)。酵母遗传学方法:冷泉港实验室课程手册。普莱恩维尤:冷泉港实验室。

7.     Klinge, S.Voigts-Hoffmann, F.Leibundgut, M.Arpagaus, S. Ban, N. (2011)与起始因子 6 复合的真核 60S 核糖体亚基的晶体结构。科学334(6058)941-948

8.     Liu, C.Apodaca, J.Davis, LE Rao, H.2007 年)。野生型酵母酿酒酵母细胞中的蛋白酶体抑制生物技术422):158160162

9.     Sambrook, J. Russell, DW (2006)蛋白质的 SDS-聚丙烯酰胺凝胶电泳。CSH 协议2006(4)pdb.prot4540

10.  Schneider-Poetsch, T., Ju, J., Eyler, DE, Dang, Y., Bhat, S., Merrick, WC, Green, R., Shen, B. Liu, JO (2010)放线菌酮和拉克米霉素对真核翻译延伸的抑制。Nat Chem Biol 6(3): 209-217

11.  Schuster, R., Anton, V., Simoes, T., Altin, S., den Brave, F., Hermanns, T., Hospenthal, M., Komander, D., Dittmar, G., Dohmen, RJ Escobar-Henriques, M. (2020)GTPase 构象开关对通过丝裂融合蛋白泛素化进行膜融合的双重作用。生命科学联盟3(1)e201900476

12.  Schuster, R.Simões, T.den Brave, F. Escobar-Henriques, M.2018 年)。低丰度泛素化形式的分离和可视化。生物方案8(22)e3081

13.  Simões, T.Schuster, R.den Brave, F. Escobar-Henriques, M.2018 年)。Cdc48 调节对线粒体融合至关重要的去泛素化酶级联反应。 Elife 7e30015

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Copyright Buntenbroich et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Buntenbroich, I., Simões, T. and Escobar-Henriques, M. (2021). Analysis of Protein Stability by Synthesis Shutoff. Bio-protocol 11(22): e4225. DOI: 10.21769/BioProtoc.4225.
  2. Simões, T., Schuster, R., den Brave, F. and Escobar-Henriques, M. (2018). Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion. Elife 7: e30015.
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