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Jun 2018

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Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network
反面高尔基体网状结构中货物蛋白包装成囊泡的无细胞重组   

Xiao  TangXiao Tang*Feng  YangFeng Yang*Yusong   GuoYusong Guo  (*共同第一作者)
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Abstract

Protein sorting at the trans Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. The protocol describes how to perform the vesicle formation assay, how to isolate vesicles and how to detect cargo proteins in vesicles. This reconstitution assay can be used to quantitatively measure the efficiency of the packaging of a specific cargo protein into transport vesicles at the TGN under specific experimental conditions.

Keywords: TGN (反面高尔基体网状结构), Reconstitution (重构), Budding (出芽), Cargo sorting (货物分选), Vesicles (囊泡), Secretion (分泌)

Background

The trans Golgi network (TGN) is an essential transport hub in the secretory transport pathway. To ensure the fidelity of vesicular trafficking, eukaryotic cells employ a variety of protein sorting machineries to accurately package specific cargo proteins into transport vesicles at the TGN which are then delivered to specific destinations (Guo et al., 2014). To deepen our understanding of the specificity of the TGN sorting process, it is important to develop an assay that can faithfully reconstitute the vesicle formation and cargo sorting process at the TGN. This assay can be utilized to directly and quantitatively measure the roles of a specific factor in regulating the packaging of a specific cargo protein into transport vesicles. Cell-free reconstitution of the packaging of cargo proteins into COPII vesicles from the endoplasmic reticulum (ER) has been well established (Kim et al., 2005; Kim et al., 2007; Merte et al., 2010; Yuan et al., 2018; Niu et al., 2019;). An in vitro assay that reconstitutes the release of a specific cargo protein, TGN46, into transport vesicles at the TGN has been developed (Ponnambalam et al., 1996; Wakana et al., 2012). TGN46 is mainly localized at the TGN, although it can cycle between the TGN and the plasma membrane (Ponnambalam et al., 1996). Recently, we devised an alternative vesicle budding protocol to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN (Ma et al., 2018). We performed the vesicle formation assay by incubating cells at 20 °C to accumulate newly synthesized cargo proteins at the TGN and performed the budding reaction in the presence of the GTPase defective mutant, Sar1A(H79G), to inhibit packaging of cargo proteins into Coat Protein Complex II (COPII) vesicles at the ER. Moreover, we utilized floatation to efficiently remove cytosolic proteins that are not associated with vesicles (Figure 1). Using this assay, we have reconstituted release of planar cell polarity proteins, Vangl2 and Frizzled6, from the TGN (Ma et al., 2018). Our assay indicates that the tyrosine sorting motif on Vangl2 and the polybasic sorting motif on Frizzled6 are important for packaging into vesicles (Ma et al., 2018). The GTPase defective mutant form of Arfrp1, Arfrp1 (Q79L), can inhibit the packaging of Vangl2 in vesicles in a concentration dependent manner (Ma et al., 2018).

Materials and Reagents

  1. Razor blade
  2. 10 cm cell and tissue culture dishes (Biofil, catalog number: TCD-010100 )
  3. 15 ml centrifuge tubes (Biofil, catalog number: CFT-011150 )
  4. Falcon® 50 ml centrifuge tubes (Corning, catalog number: 352070 )
  5. 1,000 μl tips (USA Scientific, catalog number: 11112021 )
  6. 200 μl tips (Axygen, catalog number: T200-Y )
  7. 0.5-10 μl tips (Axygen, catalog number: T300 )
  8. 1.5 ml microtubes (Axygen, catalog number: 20220415 )
  9. Axygen® 1.5 ml Maxymum recovery® microcentrifuge tube (low retention) (Corning, Axygen®, catalog number: MCT-150-L-C )
  10. Polypropylene copolymer ultracentrifuge tube (2.2 ml capacity volume, Hitachi Koki S300536A)
  11. Polycarbonate tubes, 0.5 ml capacity volume (Beckman Coulter, catalog number: 343776 )
  12. 200 μl gel loading tips (Thermo Fisher Scientific, catalog number: 010-Q )
  13. Paragon® Disposable Sterile Blades (Medicom, catalog number: 90010-10 )
  14. BemisTM ParafilmTM M Laboratory Wrapping Film (Thermo Fisher Scientific, catalog number: 13-374-12 )
  15. Immobilon®-P transfer membrane PVDF 0.45 μm (Merck, catalog number: IPVH00010 )
  16. Corning 1 L filter system 0.22 μm (Corning, catalog number: 431098 )
  17. Sprague-Dawley rats
  18. Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, catalog number: 12800082 )
  19. Opti-MEM (Gibco, catalog number: 31985070 )
  20. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  21. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 10270106 )
  22. Penicillin streptomycin (Thermo Fisher Scientific, catalog number: 15140122 )
  23. Polyethylenimine (Polysciences, catalog number: 23966-1 )
  24. Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  25. Liquid nitrogen
  26. 0.25% trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200056 )
  27. OptiPrepTM density gradient medium (Sigma-Aldrich, catalog number: D1556 )
  28. Brilliant blue R (Sigma-Aldrich, catalog number: B0149 )
  29. 2-Mercaptoethanol (βME) (Sigma-Aldrich, catalog number: M6250 )
  30. 3-color regular range protein marker (Genefist, catalog number: GF6616 )
  31. Blotting-Grade Blocker (Bio-Rad Laboratories, catalog number: 1706404 )
  32. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S8032 )
  33. SuperSignalTM west pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, catalog number: 34580 )
  34. HEPES (Acros Organics, catalog number: 172571000 )
  35. Potassium chloride (KCl) (VWR Chemicals BDH®, catalog number: 2576 )
  36. Acetic acid, potassium salt (KOAc) (Fisher Scientific, catalog number: 127082 )
  37. Magnesium acetate tetrahydrate (Mg(OAc)2) (Sigma-Aldrich, catalog number: M0631 )
  38. D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  39. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 151213 )
  40. Bromophenol blue
  41. Tris (Affymetrix, catalog number: 75825 )
  42. Glycine
  43. 40% Acrylamide/Bis Solution (Bio-Rad Laboratories, catalog number: 1610148 )
  44. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678 )
  45. N,N,N′,N′-Tetramethylethylenediamine (Sigma-Aldrich, catalog number: T7024 )
  46. Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) (VWR Chemicals BDH®, catalog number: 1514L )
  47. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  48. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  49. Glycerol (Sigma-Aldrich, catalog number: G7757 )
  50. Triton® X-100 (Sigma-Aldrich, catalog number: X100 )
  51. Tween® 20 (Sigma-Aldrich, catalog number: P1379 )
  52. Protease inhibitor cocktail tablets (Roche, catalog number: 0 5056489001 )
  53. DL-dithiothreitol (Sigma-Aldrich, catalog number: D9779 )
  54. Digitonin (Sigma-Aldrich, catalog number: D141 )
  55. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  56. Creatine phosphate (Roche, catalog number: 10621722001 )
  57. Creatine kinase (Roche, catalog number: 10736988001 )
  58. Adenosine 5'-triphosphate (ATP) (Sigma-Aldrich, catalog number: 2383 )
  59. Guanosine 5'-Triphosphate Disodium Salt (GTP) (FUJIFILM Wako Pure, catalog number: 073-03113 )
  60. Methanol (Scharlab, catalog number: 0 949 )
  61. Trypsin inhibitor from glycine max (soybean) (Sigma-Aldrich, catalog number: T9003 )
  62. HyClone® trypan blue solution (GE Healthcare, HyCloneTM, catalog number: SV30084 )
  63. Antibodies
    1. Rabbit anti SEC22B was a gift from Prof. Randy Schekman (University of California, Berkeley, CA, USA), and it was used at 1:2,000
    2. Sheep anti TGN46 (Bio-Rad Laboratories, catalog number: AHP500G ), and it was used at 1:2,000
    3. Rabbit anti HA (Cell signaling technology, catalog number: 3724 ), and it was used at 1:2,000
    4. Horseradish peroxidase (HRP)-coupled sheep antibodies against rabbit IgG (GE Healthcare, catalog number: NA934 ), and it was used at 1:5,000
    5. Anti-sheep IgG–Peroxidase antibody (Sigma-Aldrich, catalog number: A3415 ), and it was used at 1:5,000
  64. Buffer solutions (see Recipes)
    1. KHM buffer (1×)
    2. KHM buffer (2×)
    3. KHM buffer (10×)
    4. SDS-PAGE protein loading buffer (5×)
    5. Transfer buffer
    6. SDS-PAGE running buffer
    7. 5% blotting-grade blocker
    8. PBS
    9. PBST
    10. Buffer E
    11. Lysis buffer
  65. Stock solutions (see Recipes)
    1. 10× PBS, pH 7.4
    2. SDS-PAGE running buffer (10×)
    3. 1 M KOAc
    4. 1 M HEPES-KOH, pH 7.2
    5. 1 M Mg(OAc)2
    6. 1.5 M Tris-HCl, pH 8.8
    7. 1 M Tris-HCl, pH 6.8
    8. 10% SDS
    9. 10% APS
    10. 3% NaN3
    11. 40 mg/ml digitonin
    12. 100x Protease inhibitors
    13. 500 mM DTT
    14. ATP regeneration system (ATP r.s.)
    15. 10 mM GTP
    16. 1 mg/ml polyethylenimine

Equipment

  1. Small beaker
  2. Dissection scissors
  3. -80 °C freezer
  4. Drill (Craftsman 3/8 inch professional electric drill) (Craftsman, model: 315.26946 0 or equivalent)
  5. Dounce homogenizer (Kimble® 886000-0024 Kontes® 45 ml Potter-Elvehjem Tissue Grinder with PTFE Pestle and Unground Glass Tube, Size: 24, Manufacturer Part No: KIM-886000-0024)
  6. Eppendorf® 5418R centrifuge, refrigerated, with rotor FA-45-18-11 and rotor lid (Eppendorf®, model: 5418R , catalog number: 5401000013)
  7. Beckman Coulter high speed centrifuge, with rotor JA-25.50 and rotor lid (Beckman Coulter, model: Avanti® J-E, catalog number: 369001)
  8. Hitachi ultracentrifuge with T-865 rotor and rotor lid (Hitachi, catalog number: S99978101 )
  9. Light microscope with a 10× or 20× objective (any simple or compound light microscope is fine)
  10. Hitachi Koki himac CS150NX micro ultracentrifuge with S55S-2080 rotor, S120A3-2061 rotor and rotor lids (Hitachi, catalog number: HK-CS150NX )
  11. Barnstead Thermolyne (Thermo Fisher, model: DB16520-26 catalog number: 05852)
  12. Elite dry bath incubator (Major Science, model: EL-02 catalog number: 95070)
  13. Eppendorf® 5804R centrifuge with rotor A-4-44 (Eppendorf®, model: 5804R , catalog number: 5805000327)
  14. Cell disruptor (Disruptor Genie®, catalog number: SI-D268 )
  15. ChemiDocTM MP Imaging System (Bio-Rad Laboratories, model: ChemiDocTM MP)

Software

  1. ImageLab software v4.0
  2. ImageJ

Procedure

Note: All procedures are performed on ice and all centrifugations are performed at 4 °C unless otherwise stated.

  1. Preparation of cytosol from Rat Liver
    1. Dissect out livers from a couple of large Sprague-Dawley rats. Each liver typically weighs around 15 g. Usually the final yield per liver is around 15 ml of cytosolic fraction with a protein concentration of 30 mg/ml.
      Note: An application to use large Sprague-Dawley rats needs to be submitted and approved by the University of Committee on Use and Care of Animals (UCUCA).
    2. Upon dissection, place each liver in chilled 50 ml Corning tube, rinse three times with cold PBS (around 25 ml per wash).
    3. Weigh livers, then cut up into a mush with razor blade and dissection scissors on an ice-cold Petri dish. Add 2 ml cold Buffer E per 1 g liver (Recipe A1).
    4. Homogenize in large Dounce with drill press in batches (around 1 liver per batch) in cold room. 5 strokes with medium-fitting pestle, then 5 strokes with tight-fitting pestle. To be careful, wear goggles. After homogenization, rat livers will be homogenized to tiny pieces and large fragments of rat livers will not be clearly detected.
    5. Centrifuge the whole homogenate in 50 ml tubes at 1,000 x g for 10 min at 4 °C in Eppendorf® 5418R refrigerated centrifuge using FA-45-18-11 rotor. Collect supernatant and discard pellet.
    6. Centrifuge the supernatant at 18,459 x g for 20 min at 4 °C in Beckman Coulter high speed centrifuge (Beckman Coulter, Avanti® J-E) using JA-25.50 rotor. Collect supernatant and discard pellet.
    7. Centrifuge the supernatant at 125,171 x g for 1 h at 4 °C in Hitachi ultracentrifuge (Hitachi ultracentrifuge) using T-865 rotor.
    8. Take the deep red fraction (avoid pellet and the floating fat layer) and repeat high speed spin 2 or 3 times.
    9. Determine protein concentration by the Bradford assay and aliquot in 100 μl and 1 ml amounts.
      Note: a concentration range of 18-30 mg/ml is considered as a successful extraction.
    10. Snap freeze aliquots in liquid nitrogen and store at -80 °C. Avoid repeated freeze-thaw cycles.
      Note: The centrifugation steps to prepare rat liver cytosol are shown in Figure 1.


      Figure 1. The centrifugation steps to prepare rat liver cytosol

  2. Preparation of donor membranes (DM) from cultured HEK 293T cells (prepare on the day of the reaction, Figure 2)


    Figure 2. Schematic overview of the assay to reconstitute vesicular release from the TGN. The preparations of semi-intact cells as donor membranes (DM) for the budding reaction and the procedures of the vesicle formation assay and isolation of vesicles produced from semi-intact cells were described in Procedures B and C respectively. Efficiency of the packaging of cargos into vesicles were measured by immunoblotting as described in Procedure D (this Figure is revised from Ma et al., 2018). The DM are also needed for immunoblotting.

    1. Culture two 10 cm plates of HEK 293T cells in GIBCO Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS), 10 milliunits/ml of penicillin, and 0.1 mg/ml of streptomycin at 37 °C in a cell culture CO2 incubator providing 5% CO2 and around 95% relative humidity. The cells will be grown to 80-100% confluent on the day of the experiment.
      Note: Transfect plasmids encoding cargo proteins 24 h before the day of experiment if necessary.
    2. Aspirate media from two 10 cm plates of HEK 293T cells.
    3. Wash the cells with 5 ml PBS each plate for 2 times.
    4. Add 10 ml Opti-MEM containing 10% FBS into each plate, seal the plate with parafilm and flow it at 20 °C water bath for 2 h.
    5. Wash the cells with 5 ml PBS for 2 times for each plate.
    6. Add 1 ml 0.25% trypsin to each plate and incubate at RT for 2 min.
    7. Collect cells from each plate with 6 ml PBS buffer into one15 ml Falcon tube.
    8. Add 25 μl 10 mg/ml trypsin inhibitor to each tube and mix well.
    9. Centrifuge at 300 x g for 3 min at 4 °C. Aspirate the supernatant and resuspend the cells in 4 ml 1× KHM buffer (Recipe A1) in each tube.
    10. Add 4 μl digitonin (stock 40 mg/ml in DMSO) to the cells so that final concentration is 40 μg/ml. Mix well and incubate on ice for 5 min.
    11. Add 8 ml 1× KHM buffer to the cells in each tube, invert the tube and immediately pellet the cells at 300 x g for 3 min at 4 °C.
    12. Aspirate the supernatant, resuspend the cell pellet in 1 ml 1× KHM buffer in each tube and transfer to a 1.5 ml microcentrifuge tube.
      Note: To check if permeabilization is working or not, add 3 μl of cells to 3 μl trypan blue, carefully lay a cover slip over the sample, then check percentage of permeabilized cells under a light microscope with a 16× objective. 100% of cells should be permeabilized at this stage. The nucleus is blue and the intact ER is brown. If the permealization is not working well, a freshly made solution of digitonin is recommended to perform the experiments.
    13. Incubate the cells on ice for 5 min, pellet the cells at 10,000 x g for 5 s at 4 °C and aspirate the supernatant. Resuspend the pellet in 130 μl 1× KHM buffer in each tube and combine the suspensions of semi-intact cells into one 1.5 ml microcentrifuge tube.

  3. Reconstitution of the release of vesicles from the TGN
    1. In low retention tubes, assemble vesicle budding reactions by adding ingredients in Table 1. Each 100 μl reaction contains ATP regeneration system (1 mM ATP, 40 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase in KHM buffer), 0.2 mM GTP, 1 μg Sar1A H79G protein (a GTPase defective mutant version of Sar1A) and rat liver cytosol (4 mg/ml).
      Note: Sar1A H79G protein is a C-terminal His-tagged protein purified from E. coli. Add KHM buffer, nucleotides and Sar1A H79G protein first. Mix well by pipetting and briefly centrifuge to collect liquid at the bottom of each tube. Then add semi-intact cells and mix by pipetting up and down gently until homogenous. Add rat liver cytosol last and mix by gentle pipetting.

      Table 1. The ingredients added in the TGN vesicle formation assay

      Note: Each column represents a single experimental group. Each row represents a component of the reaction and the volume added to the reaction in μl. The total volume of the reaction is 500 μl. The experimental group without nucleotide is used to monitor the level of cytosolic proteins that are diffused to the vesicle fraction after centrifugation and to monitor the level of cargo proteins that are in the vesicle fraction independent of GTP and ATP. The experimental group performed in the presence of GMPNP, a non-hydrolysable analog of GTP, is to monitor the level of cytosolic and cargo proteins that are present in the vesicle fraction independent of GTP hydrolysis. Sar1A (H79G) is defective in GTP hydrolysis and inhibits packaging of cargo proteins into COPII vesicles. This protein is added to the budding reaction to inhibit COPII-mediated vesicle formation from the ER.

    2. Incubate reactions at 32 °C for 1 h.
    3. Transfer reactions to ice to stop reaction after incubation.
    4. Centrifuge at 14,000 x g for 20 min at 4 °C in Eppendorf® 5418R refrigerated centrifuge using FA-45-18-11 rotor to remove cell debris and large membranes.
    5. Mix supernatant with cold 60% (w/v) OptiPrepTM and 10× KHM buffer (Recipe A3) to reach final concentration at 35% (w/v) OptiPrepTM in 1× KHM buffer.
      Note: The OptiPrepTM gradient is a 60% (w/v) solution of iodixanol in water (sterile).
    6. Transfer mixed sample to the bottom of polypropylene copolymer ultracentrifuge tube (2.2 ml capacity volume, Hitachi Koki S300536A) as the bottom layer.
    7. Overlay with 30% (w/v) OptiPrepTM in 1× KHM buffer [prepared by mixing equal volume of 60% OptiPrepTM and 2× KHM buffer (Recipe A2)] by slowly pipetting against the wall using a gel-loading tip to the ultracentrifuge tube as the medium layer until the tube is almost filled with the whole sample (bottom layer + medium layer).
      Note: An interphase should be observed between 30% OptiPrepTM and the supernatant OptiPrepTM mixture at this stage.
    8. Overlay with 50 μl 1× KHM buffer at the top layer by pipetting slowly using a gel-loading tip.
      Note: An interphase should be observed between 1× KHM and 30% OptiPrepTM in 1× KHM.
    9. Centrifuge the ultracentrifuge tubes containing the sample at 100,000 x g at 4 °C for 90 min at the Acceleration Setting (Accel) 3 and Decel 4 in Hitachi Koki himac CS150NX micro ultracentrifuge (Hitachi) using S55S-2080 rotor. After centrifugation, the released vesicles will be concentrated at the interface between 1x KHM and 30% OptiPrepTM in 1× KHM.
    10. Collect vesicles by taking 200 μl supernatant from the top after centrifugation. Transfer the supernatant to thick wall polycarbonate tubes (Beckman Coulter, 0.5 ml capacity volume, 343776 ). Add 300 μl 1x KHM buffer to dilute samples by gently pipetting.
    11. Centrifuge at 100,000 x g at 4 °C for 30 min in Hitachi Koki himac CS150NX micro ultracentrifuge (Hitachi) using S120AT3-2061 rotor to spin down vesicles.

  4. Immunoblot
    Perform standard immunoblotting procedure by the following steps:
    1. Add 20 μl 1× SDS-PAGE protein loading buffer at the bottom of the tube, vortex 8 min to dissolve the vesicles in loading buffer using the cell disruptor (Disruptor Genie®).
    2. Add DM to 20 μl lysis buffer (Recipe A11) and incubate on ice for 30 min.
    3. Centrifuge at 14,000 x g for 5 min at 4 °C and collect the supernatant.
    4. Add 5× SDS-PAGE protein loading buffer (Recipe A4) into the supernatant to reach a final concentration of 1× SDS-PAGE protein loading buffer.
    5. Incubate both DM sample and high spin pellet (HSP) samples at 55 °C for 30 min.
    6. Load the DM sample and HSP samples onto a 15 wedged well 10-15% gel according to the cargo protein size.
    7. Run SDS-PAGE at constant 20 mA until dye runs out of the gel (about 70 min) at RT.
    8. Transfer protein onto a PVDF membrane at constant 0.3 A for 1-2 h at 4 °C.
    9. Block the PVDF membrane with 5% blotting-Grade Blocker in PBST (Recipe A7) for 30 min at RT.
    10. Incubate the PVDF membrane with primary antibodies at RT for 1.5-2 h or at 4 °C overnight.
    11. Wash with PBST 3 × 5 min.
    12. Incubate with secondary antibodies conjugated with HRP at RT for 1 h.
    13. Wash with PBST 3 × 5 min.
    14. Incubate with the HRP substrate ECL plus and image on a ChemiDocTM Imaging System with ImageLab software v4.0.

Data analysis

  1. Export immunoblot images from ImageLab software v4.0 as .tif files.
  2. Use Photoshop and Adobe Illustrator® to process images (Figure 3).


    Figure 3. Reconstitution of packaging of Frizzled6 into vesicles from the TGN. COS7 cells were transfected with HA-Frizzled6. On day 1 after transfection, TGN vesicle release reaction was performed using the indicated reagents described in Table 1. The top fraction after flotation was analyzed by immunoblotting with the indicated antibodies. TGN46 and Sec22 are used as positive controls to monitor TGN export and ER export respectively (Republished from Ma et al., 2018).

Recipes

  1. Buffer solutions
    1. 1× KHM (1 L)
      1. Add 5 ml 1 M Mg(OAc)2, 20 ml 1 M HEPES-KOH, pH 7.2 and 110 ml 1 M KOAc to 0.5 L ddH2O
      2. Adjust volume to 1 L
      3. Filter the buffer
      4. Store at 4 °C
    2. 2× KHM (1 L)
      1. Add 10 ml 1 M Mg(OAc)2, 40 ml 1 M HEPES-KOH, pH 7.2 and 220 ml 1 M KOAc to 0.5 L ddH2O
      2. Adjust volume to 1 L
      3. Filter the buffer
      4. Store at 4 °C
    3. 10× KHM (1 L)
      1. Add 50 ml 1 M Mg(OAc)2 and 200 ml 1 M HEPES-KOH, pH 7.2 to 0.5 L ddH2O
      2. Dissolve 108 g KOAc
      3. Adjust volume to 1 L
      4. Filter the buffer
      5. Store at 4 °C
    4. 5× SDS-PAGE protein loading buffer (50 ml)
      1. Dissolve 3.75 g SDS, 93.75 mg bromophenol blue in a small beaker with 12.5 ml 1 M Tris pH6.8 with constant stirring
      2. Mix in 25 ml glycerol
      3. Adjust volume to 37.5 ml with ddH2O
      4. Aliquot to 750 μl/tube and store at -20 °C
      5. Add 250 μl βME to an aliquot fresh before use
        Note: The final concentration of each reagents in 5× SDS-PAGE protein loading buffer: 7.5% SDS, 50% Glycerol, 250 mM Tris-HCl and 0.19% bromophenol blue, 25% βME (v/v).
    5. Transfer buffer (2 L)
      1. Dissolve 6.03 g Tris and 28.8 g glycine in 1.5 L ddH2O (final concentration: 25 mM Tris, 192 mM glycine)
      2. Add 200 ml Methanol (final concentration: 10%)
      3. Add ddH2O to 2 L
      4. Store at 4 °C
    6. SDS-PAGE running buffer (1 L)
      Add 100 ml 10× SDS-PAGE running buffer in 900 ml ddH2O
    7. 5% blotting-grade blocker
      Dissolve 5% blotting-grade blocker (w/v) in PBST
    8. PBS (1 L)
      Add 100 ml 10× PBS in 900 ml 10× PBS
    9. PBST
      0.1% TWEEN® 20 (v/v) in 1× PBS
    10. Buffer E (1 L)
      1. Add 50 ml 1 M HEPES-KOH, pH 7.2, 70 ml 1 M KOAc and 0.5 ml 1 M Mg(OAc)2 in 800 ml ddH2O
      2. Dissolve 45.54 g sorbitol and 1.9 g potassium EGTA in the buffer
      3. Adjust volume to 1 L
      4. Filter the buffer
      5. Aliquot to small volume and store at -80 °C
      6. Add protease inhibitor at 1× concentration and 5 mM DTT before use
    11. Lysis buffer
      Add 0.5% Triton® X-100, 1× protease inhibitor and 1 mM DTT in KHM buffer

  2. Stock solutions
    1. 10× PBS, pH 7.4 (2 L)
      1. Dissolve 160 g of NaCl, 4 g KCl, 28.8 g Na2HPO4 and 4.8 g KH2PO4 in 1.9 L ddH2O
      2. Adjust pH to 7.4 with NaOH
      3. Add ddH2O to 2 L
      4. Sterilize by autoclaving and store at RT
    2. 10× SDS-PAGE running buffer (1 L)
      1. Dissolve 30.3 g Tris, 144.4 g glycine and 10 g SDS in 1 L ddH2O
      2. Store at RT
    3. 1 M KOAc (1 L)
      1. Dissolve 98.14 g potassium acetate 1 L ddH2O
      2. Sterilize by autoclaving and store at RT
    4. 1 M HEPES-KOH, pH 7.2 (1 L)
      1. Dissolve 238.3 g of HEPES in 0.5 L ddH2O
      2. Adjust pH to 7.2 with KOH
      3. Add ddH2O to 1 L
      4. Store at RT
    5. 1 M Mg(OAc)2 (1 L)
      1. Dissolve 155.41 g Mg(OAc)2 in 1 L ddH2O
      2. Sterilize by autoclaving and store at RT
    6. 1.5 M Tris-HCl, pH 8.8 (1 L)
      1. Dissolve 181.7 g of Tris in 0.8 L ddH2O
      2. Adjust pH to 8.8 with HCl
      3. Add ddH2O to 1 L
      4. Sterilize by autoclaving and store at RT
    7. 1 M Tris-HCl, pH 6.8 (1 L)
      1. Dissolve 121.1 g of Tris in 0.8 L ddH2O
      2. Adjust pH to 6.8 with HCl
      3. Add ddH2O to 1 L
      4. Sterilize by autoclaving and store at RT
    8. 10% SDS (100 ml)
      1. Dissolve 10 g of SDS in 90 ml ddH2O
      2. Store at RT
    9. 10% APS (50 ml)
      1. Dissolve 5 g of SDS in 50 ml ddH2O
      2. Store at 4 °C
    10. 3% NaN3 (50 ml)
      1. Dissolve 1.5 g of NaN3 in 50 ml ddH2O
      2. Store at RT
    11. 40 mg/ml digitonin (10 ml)
      1. Dissolve 400 mg digitonin in 10 ml ddH2O
      2. Aliquot in volumes of 30 μl, store at -20 °C
    12. 100× protease inhibitors (1 ml)
      1. Dissolve 2 protease inhibitor cocktail tablet in 1 ml ddH2O
      2. Aliquot in volumes of 30 μl, store at -20 °C
    13. 500 mM DTT (1 ml)
      1. Dissolve 0.077 g DL-dithiothreitol in 1 ml ddH2O
      2. Aliquot in volumes of 30 μl, store at -20 °C
    14. ATP regeneration system (20 ml)
      1. Dissolve 2.04 g creatine phosphate, 40 mg creatine kinase, 101.44 mg ATP in 20 ml 1× KHM
      2. Aliquot in small volumes and store at -80 °C
    15. 10 mM GTP (20 ml)
      1. Dissolve 113.43 mg ATP in 20 ml 1× KHM
      2. Aliquot in small volumes and store at -80 °C
    16. 1 mg/ml polyethylenimine (100 ml)
      1. Dissolve 0.1 g polyethylenimine in 100 ml ddH2O
      2. Filtered by 0.22 μm filter
      3. Aliquot in small volumes and store at -80 °C

Acknowledgments

This protocol was modified from previous work described in previous reports (Kim et al., 2005; Yuan et al., 2017; Ma et al., 2018; Niu et al., 2019). This work was supported by the Hong Kong Research Grants Council Grants 26100315, 16102218, 16103319 and 16101116 to Y.G. Y.G. was a previous HHMI postdoctoral research associate during the period of 2009-2013.

Competing interests

The authors declare no conflict of interest or competing interests.

References

  1. Guo, Y., Sirkis, D. W. and Schekman, R. (2014). Protein sorting at the trans-Golgi network. Annu Rev Cell Dev Biol 30: 169-206.
  2. Kim, J., Hamamoto, S., Ravazzola, M., Orci, L. and Schekman, R. (2005). Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles. J Biol Chem 280(9): 7758-7768.
  3. Kim, J., Kleizen, B., Choy, R., Thinakaran, G., Sisodia, S. S. and Schekman, R. W. (2007). Biogenesis of gamma-secretase early in the secretory pathway. J Cell Biol 179(5): 951-963.
  4. Ma, T., Li, B., Wang, R., Lau, P. K., Huang, Y., Jiang, L., Schekman, R. and Guo, Y. (2018). A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network. J Biol Chem 293(22): 8410-8427.
  5. Merte, J., Jensen, D., Wright, K., Sarsfield, S., Wang, Y., Schekman, R. and Ginty, D. D. (2010). Sec24b selectively sorts Vangl2 to regulate planar cell polarity during neural tube closure. Nat Cell Biol 12(1): 41-46; sup pp 41-48.
  6. Niu, L., Ma, T., Yang, F., Yan, B., Tang, X., Yin, H., Wu, Q., Huang, Y., Yao, Z. P., Wang, J., Guo, Y. and Hu, J. (2019). Atlastin-mediated membrane tethering is critical for cargo mobility and exit from the endoplasmic reticulum. Proc Natl Acad Sci U S A 116(28): 14029-14038.
  7. Ponnambalam, S., Girotti, M., Yaspo, M. L., Owen, C. E., Perry, A. C., Suganuma, T., Nilsson, T., Fried, M., Banting, G. and Warren, G. (1996). Primate homologues of rat TGN38: primary structure, expression and functional implications. J Cell Sci 109 (Pt 3): 675-685.
  8. Wakana, Y., van Galen, J., Meissner, F., Scarpa, M., Polishchuk, R. S., Mann, M. and Malhotra, V. (2012). A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface. EMBO J 31(20): 3976-3990.
  9. Yuan, L., Baba, S., Bajaj, K. and Schekman, R. (2017). Cell-free generation of copii-coated Procollagen I carriers. Bio-protocol 7(22): e2450.
  10. Yuan, L., Kenny, S. J., Hemmati, J., Xu, K. and Schekman, R. (2018). TANGO1 and SEC12 are copackaged with procollagen I to facilitate the generation of large COPII carriers. Proc Natl Acad Sci U S A 115(52): E12255-E12264.

简介

[摘要] 反式高尔基体网络(TGN)上的蛋白质分选在将新合成的蛋白质靶向其特定目的地方面起着重要作用。该提议的目的是利用大鼠肝细胞溶质,半完整的哺乳动物细胞和核苷酸,在TGN处将非高尔基驻留的货物蛋白重新包装成囊泡。该协议描述了如何进行囊泡形成测定,如何分离囊泡以及如何检测囊泡中的货物蛋白。该重构测定法可用于定量测量在特定实验条件下将特定货物蛋白包装到TGN的运输小泡中的效率。

[背景] 的反式高尔基体网络(TGN)是在分泌运送路径的必要的交通枢纽。为了确保水泡运输的保真度,真核细胞利用各种蛋白质分选设备将特定的货物蛋白质准确地包装到TGN的运输小泡中,然后运至特定的目的地(Guo 等人,2014)。为了加深我们对TGN分选过程特异性的理解,重要的是开发一种能够忠实地重构TGN囊泡形成和货物分选过程的分析方法。该测定法可用于直接和定量地测量特定因子在调节特定货物蛋白包装到运输小泡中的作用。从内质网(ER)将货物蛋白包装到COPII囊泡中的无细胞重构已得到很好的建立(Kim 等,2005; Kim 等,2007; Merte 等,2010; Yuan 等。,2018;Niu 等,2019;)。已经开发出一种体外测定法,其在TGN处重构特定货物蛋白TGN46在运输小泡中的释放(Ponnambalam 等,1996;Wakana 等,2012)。TGN46主要位于TGN,尽管它可以在TGN和质膜之间循环(Ponnambalam 等,1996)。最近,我们设计了另一种囊泡出芽方案,以在TGN上将非高尔基驻留的货物蛋白包装成囊泡( Ma等,2018)。我们通过在20°C下孵育细胞以在TGN上积累新合成的货物蛋白来进行囊泡形成试验,并在存在GTPase缺陷型突变体Sar1A(H79G)的情况下进行出芽反应,以抑制货物蛋白包装到外壳蛋白中ER处的复合物II(COPII)囊泡。此外,我们利用浮选法有效地去除了与囊泡无关的胞质蛋白(图1)。使用该测定法,我们从TGN重构了平面细胞极性蛋白Vangl2和Frizzled6的释放(Ma 等,2018)。我们的测定表明,Vangl2上的酪氨酸分选基序和Frizzled6上的多元分选基序对于包装成小泡很重要(Ma 等,2018)。Arfrp1的GTPase缺陷型突变体Arfrp1(Q79L)可以以浓度依赖的方式抑制Vangl2在囊泡中的包装(Ma et al。,2018)。

关键字:反面高尔基体网状结构, 重构, 出芽, 货物分选, 囊泡, 分泌

材料和试剂


 


剃须刀
10厘米细胞和组织培养皿(Biofil ,目录号:TCD-010100)
15 ml离心管(Biofil ,目录号:CFT-011150)
鹘? 50ml离心管中(Corning,目录号:352070)
1000点微升的提示(美国科学,目录号:11112021)
200点微升的提示(爱思进,目录号:T200-Y)
0.5-10 微升提示(爱思进,目录号:T300)
1.5 ml 微管(Axygen ,目录号:20220415)
爱思进? 1.5毫升Maxymum 恢复? 离心管(低保留)(Corning公司,爱思进? ,目录号:MCT-150-LC)
聚丙烯共聚物超速离心管(容量为2.2 ml,日立工机S300536A)
聚碳酸酯管,容量为0.5 ml (Beckman Coulter,目录号:343776)
200个微升凝胶放入提示(赛默飞世尔科技,产品目录号:010-Q)
帕拉贡? 一次性使用无菌刀片(穿衣,目录号:90010-10)
Bemis TM Parafilm TM M实验室包装膜(Thermo Fisher Scientific,目录号:13-374-12)
的Immobilon ? -P转移膜PVDF 0.45 微米(默克,目录号:IPVH00010)
康宁1升过滤器系统0.22 微米(康宁,目录号:431098)
Sprague-Dawley大鼠
Dulbecco的改良Eagle培养基(DMEM)(Thermo Fisher Scientific,目录号:12800082)
Opti-MEM(Gibco,目录号:31985070)
碳酸氢钠(Sigma-Aldrich,目录号:S5761)
胎牛血清(FBS)(Thermo Fisher Scientific,目录号:10270106)
青霉素链霉素(Thermo Fisher Scientific,目录号:15140122)
聚乙烯亚胺(Polysciences ,目录号:23966-1)
Bio-Rad蛋白测定染料试剂浓缩液(Bio-Rad实验室,目录号:5000006)
液氮
0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,目录号:25200056)
OptiPrep TM 密度梯度介质(Sigma-Aldrich,目录号:D1556)
艳蓝色R(Sigma-Aldrich,目录号:B0149)
2-巯基乙醇(βME)(Sigma-Aldrich,目录号:M6250)
3色常规蛋白质标记物(Genefist ,目录号:GF6616)
印迹等级阻滞剂(Bio-Rad实验室,目录号:1706404)
钠叠氮化物(NAN 3 )(Sigma-Aldrich公司,目录号:S8032)
SuperSignal TM 西微微PLUS化学发光底物(Thermo Fisher Scientific,目录号:34580)
HEPES(Acros Organics,目录号:172571000)
氯化钾(KCl的)(VWR化学BDH ? ,目录号:2576)
36. 乙酸钾盐(KOAc )(Fisher Scientific,目录号:127082)   
四水合醋酸镁(Mg (OAc )2 )(Sigma-Aldrich,目录号:M0631)
D-山梨糖醇(Sigma-Aldrich,目录号:S1876)
十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:151213)
溴酚蓝
Tris(Affymetrix,目录号:75825)
甘氨酸
40%丙烯酰胺/双解溶液(Bio-Rad Laboratories,目录号:1610148)
过硫酸铵(APS)(Sigma-Aldrich,目录号:A3678)
45. N ,N,N ',N'- 四甲基乙二胺(Sigma-Aldrich,目录号:T7024)   
46。    磷酸二氢钠二水合物(的NaH 2 PO 4 ·2H 2 O)(VWR化学BDH ? ,目录号:1514L)
47。    磷酸氢二钾(K 2 HPO 4 )(Sigma-Aldrich,目录号:P3786)
48. 氯化钠(NaCl)(西格玛奥德里奇,目录号:S5886)   
甘油(Sigma-Aldrich,目录号:G7757)
的Triton ? X100(Sigma-Aldrich公司,目录号:X100)
吐温? 20(Sigma-Aldrich公司,目录号:P1379)
蛋白酶抑制剂鸡尾酒片剂(罗氏,目录号:05056489001)
DL-二硫苏糖醇(Sigma-Aldrich,目录号:D9779)
洋地黄皂苷(Sigma-Aldrich,目录号:D141)
二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
磷酸肌酸(罗氏(Roche),目录号:10621722001)
肌酸激酶(罗氏(Roche),目录号:10736988001)
腺苷5 ' 三磷酸(ATP)(Sigma-Aldrich公司,目录号:2383)
鸟苷5'-三磷酸二钠盐(GTP)(FUJIFILM Wako Pure,目录号:073-03113)
甲醇(Scharlab ,目录号:0949)
来自glycine max(大豆)的胰蛋白酶抑制剂(Sigma-Aldrich,目录号:T9003)
HyClone公司? 台盼蓝溶液(GE Healthcare公司,HyClone公司TM ,目录号:SV30084)
抗体
Rabbit anti SEC22B是Randy Schekman 教授(美国加利福尼亚大学伯克利分校,美国)提供的礼物,使用量为1:2,000
绵羊抗TGN46(Bio-Rad实验室,目录号:AHP500G),以1:2,000的浓??度使用
兔抗HA(细胞信号技术,目录号:3724),以1:2,000的比例使用
辣根过氧化物酶(HRP)偶联的羊抗兔IgG抗体(GE Healthcare,目录号:NA934),以1:5,000的浓??度使用
抗绵羊IgG-过氧化物酶抗体(Sigma-Aldrich,目录号:A3415),以1:5,000的浓??度使用
缓冲溶液(请参阅配方)
KHM缓冲区(1×)
KHM缓冲器(2×)
KHM缓冲器(10×)
SDS-PAGE蛋白加载缓冲液(5×)
传输缓冲区
SDS-PAGE运行缓冲区
5%印迹级别的阻滞剂
PBS
PBST
缓冲液E
裂解缓冲液
储备溶液(请参阅食谱)
10×PBS,pH 7.4
SDS-PAGE运行缓冲区(10 ×)
1 M KOAc
1 M HEPES-KOH,pH 7.2
1 M镁(OAc )2
1.5 M Tris-HCl,pH 8.8
1 M Tris-HCl,pH 6.8
10%SDS
10%APS
3%NaN 3
40 mg / ml洋地黄皂苷
100x蛋白酶抑制剂
500毫米DTT
ATP再生系统(ATP rs 。)
10 mM GTP
1毫克/毫升聚乙烯亚胺




设备


 


小烧杯
解剖剪刀
-80°C冷冻室
钻(工匠3/8英寸专业电钻)(工匠,型号315.269460 或等效)
杜恩斯匀浆器(金宝? 886000-0024 Kontes ? 45毫升Potter- Elvehjem 组织研磨聚四氟乙烯杵和非磨玻管,大小:24,制造商零件编号:KIM-886000-0024)
的Eppendorf ? 5418R离心机,冷藏,与转子FA-45-18-11和转子盖仪(Eppendorf ? ,型号:5418R,目录号:5401000013)
Beckman Coulter公司高速离心机,具有转子JA-25.50转子盖(Beckman Coulter公司,型号:阿凡提? JE,目录号:369001)
带T-865转子和转子盖的日立超速离心机(日立,目录号:S99978101)
具有10倍或20倍物镜的光学显微镜(任何简单或复合的光学显微镜都可以)
Hitachi Koki himac CS150NX微型超速离心机,带有S55S-2080转子,S120A3-2061转子和转子盖(日立,目录号:HK-CS150NX)
Barnstead Thermolyne (Thermo Fisher,型号:DB16520-26 目录编号:05852 )
精英干浴培养箱(主要科学,型号:EL-02目录编号:95070)
的Eppendorf ? 5804R离心机转子与A-4-44仪(Eppendorf ? ,型号:5804R,目录号:5805000327)
细胞破碎(否决精灵? ,目录号:SI-D268)
ChemiDoc TM MP成像系统(Bio-Rad实验室,型号:ChemiDoc TM MP)
 


软件


 


ImageLab 软件v4.0
图像
 


程序


 


注意:除非另有说明,所有步骤均在冰上进行,所有离心均在4°C下进行。


从大鼠肝脏制备细胞溶质
从几只大的Sprague-Dawley大鼠中解剖出肝脏。每个肝脏通常重约15克。通常,每个肝脏的最终产量约为15 ml胞质级分,蛋白质浓度为30 mg / ml。
注意:使用大型Sprague-Dawley大鼠的申请必须由动物使用和护理委员会(UCUCA)提交并批准。


解剖后,将每个肝脏放入冷却的50 ml康宁管中,用冷PBS冲洗3次(每次清洗约25 ml)。
称取肝脏,然后切割成一个糊状机智?刀片和清扫剪刀上的冰冷P ETRI菜。每1克肝脏添加2毫升冷缓冲液E(配方A 1 )。
在冷室中用钻床分批(每批约1块肝脏)进行大均质化。中型杵用5笔,紧实杵用5笔。请注意,戴上护目镜。均质化后,大鼠肝脏将被均质化为小块,并且不会清楚地检测到大鼠肝脏的大碎片。
离心机在50ml管中的整个匀浆以1000 ×g离心10分钟,在4℃在Eppendorf ? 5418R冷藏centrifu GE使用FA-45-18-11转子。收集上清液并弃去沉淀。
离心上清液在18459 ×g离心20分钟,在4 ℃下在Beckman Coulter公司高速离心机(Beckman Coulter公司,阿凡提? 使用JA-25.50 RO JE)TOR。收集上清液并弃去沉淀。
离心上清液在125171 ×g下1个小时,在4 ℃下我N使用T-865转子日立超速离心机(日立超速离心机)。
取深红色部分(避免沉淀和漂浮的脂肪层),并重复高速旋转2或3次。
由第测定蛋白浓度?Bradford测定和等分试样在100 μ 升和1ml的量。
注意:18-30 mg / ml的浓度范围被认为是成功提取。


将速冻等分试样迅速注入液氮中,并储存在-80 °C下。避免重复冻融循环。
注意:制备大鼠肝细胞溶质的离心步骤如图1所示。


 


D:\ Reformatting \ 2020-1-6 \ 1902842--1296郭玉松636915 \ Figs jpg \图1.jpg


图1.离心制备大鼠肝细胞溶质


 


从培养的HEK 293T细胞制备供体膜(DM)(在反应当天制备,图2)
 


D:\ Reformatting \ 2020-1-6 \ 1902842--1296郭玉松636915 \ Figs jpg \图2.jpg


图2.从TGN重构水泡释放的测定的示意图。半完整的细胞作为供体膜(DM)的出芽反应和囊泡形成测定和从半完整细胞产生的小泡的隔离的程序的制剂在过程被描述小号B和C分别。货物进入囊泡的包装效率,通过如在步骤d中描述(免疫测定吨他的图是从马订正等人,2018)。在DM 都还需要进行免疫印迹。


 


在37 °C 的细胞培养CO 2 培养箱中,在GIBCO Dulbecco改良的Eagle培养基中培养两个10 cm的HEK 293T细胞板,该培养基包含10%胎牛血清(FBS),10 milliunits / ml青霉素和0.1 mg / ml链霉素。提供5%的CO 2 和约95%的相对湿度。在实验当天,细胞将生长至80-100%融合。
注意:如有必要,转染质粒可在实验日前24小时编码货物蛋白。


从两个10 cm HEK 293T细胞板中吸出培养基。
每个板用5 ml PBS洗涤细胞2次。
在每个平板中加入10 ml含10%FBS的Opti-MEM,用石蜡膜密封平板,并在20°C水浴中流动2小时。
每个板用5 ml PBS 洗涤细胞2次。
向每个平板中加入1 ml 0. 25%胰蛋白酶,并在室温下孵育2分钟。
用6 ml PBS缓冲液从每块板中收集细胞到15 ml F alcon管中。
向每个试管中加入25μl10 mg / ml胰蛋白酶抑制剂,并充分混合。
在4°C下以300 xg离心3分钟。吸出上清液,将细胞重悬于每个试管中的4 ml 1×KHM缓冲液(配方A 1 )中。  
向细胞中加入4μl 洋地黄皂苷(在DMSO中储备40 mg / ml),使最终浓度为40μg / ml。充分混合并在冰上孵育5分钟。
在每个试管中的细胞中加入8 ml 1 ×KHM缓冲液,颠倒试管,立即在4°C下以300 xg 沉淀细胞3分钟。
吸出上清液,将细胞沉淀重悬于每个试管中的1 ml 1×KHM缓冲液中,并转移至1.5 ml微量离心管中。
注意:要检查通透性是否正常,请添加3 μ升细胞的3 μ 升台盼蓝,小心地放置在样品上盖玻片,然后检查透化的细胞的百分比在光学显微镜下以16×物镜。在此阶段应透化100 %的细胞。核为蓝色,完整的ER为棕色。如果渗透效果不佳,建议使用新鲜制备的洋地黄皂苷溶液进行实验。


在冰上孵育细胞5分钟,在10,000 xg 于4°C下沉淀细胞5 s,然后吸出上清液。重悬130沉淀微升1×KHM在每个管缓冲液和半完整细胞的悬浮液合并成一个1.5 ml离心管。
 


重建TGN释放的囊泡
在低保持管,组装囊泡通过在表1中的每个100加入配料出芽反应μ 升反应含有ATP再生系统(1毫摩尔ATP,40mM的磷酸肌酸,0.2毫克/毫升的肌酸phosphoki 在KHM缓冲器核苷酸酶),0.2 mM的GTP ,1个μ 克Sar1A H79G蛋白(GTP酶有缺陷的Sar1A突变形式)和大鼠肝胞质溶胶(4毫克/毫升)。
注意:Sar1A H79G蛋白是从大肠杆菌纯化的C端His标记蛋白。首先添加KHM缓冲液,核苷酸和Sar1A H79G蛋白。用移液器充分混合,并短暂离心以收集每个管底部的液体。然后加入半完整细胞,并轻轻上下吹打直至均匀,进行混合。最后加入大鼠肝细胞溶质,并轻轻移液混合。


 


表1. TGN囊泡形成试验中添加的成分


 


没有核苷酸


没有Sar1A(H79G)


一切


加GMPPNP


KHM缓冲器


395


340


335


335


ATP 再生系统


0


50


50


50


GTP


0


10


10


0


GMPPNP


0


0


0


10


Sar1A(H79G)(1 μ 克/ ml)的


5


0


5


5


半无效细胞


50


50


50


50


胞浆(终浓度2 mg / ml)


50


50


50


50





500


500


500


500


注意:每列代表一个实验组。每行代表反应,并加入到reactio体积的部件在正μ 升。反应的总体积为500 μ 升。而不核苷酸实验组是使用吨O监控胞质水平被离心后扩散到囊泡分数蛋白,并监测货物蛋白质是在囊泡分数独立GTP和ATP的水平。在GMPNP(一种不可水解的GTP类似物)的存在下进行的实验小组是要监测与GTP水解无关的囊泡级分中存在的胞质蛋白和货物蛋白的水平。Sar1A(H79G)在GTP水解中有缺陷,并抑制货物蛋白包装到COPII囊泡中。将该蛋白质添加到出芽反应中,以抑制ER介导COPII介导的囊泡形成。


 


将反应液在32°C下孵育1小时。
孵育后将反应转移至冰中以终止反应。
离心机以14,000 ×g离心20分钟在Eppendorf在4℃下? 5418R使用FA-45-18-11转子以除去细胞碎片的冷冻离心机d LA RGE膜。
将上清液与冷的60%(w / v)OptiPrep TM 和10x KHM缓冲液(配方A 3 )混合,以在1x KHM缓冲液中达到35%(w / v)OptiPrep TM的最终浓度。
注意:OptiPrep TM 梯度是碘克沙醇在水中的60%(w / v)溶液(无菌)。


将混合样品转移到聚丙烯共聚物超速离心管(容量为2.2 ml,Hitachi Koki S300536A)的底部作为底层。
通过使用凝胶加载尖端在墙上缓慢吸移,在1 × KHM缓冲液中覆盖30%(w / v)OptiPrep TM [ 通过将等体积的60%OptiPrep TM 和2 × KHM缓冲液(配方A 2混合)制备] 作为中层进入超速离心管,直到该管几乎充满了整个样品(底部层+中等层)。
注意:在此阶段,应观察到30%OptiPrep TM 和上清液OptiPrep TM 混合物之间存在相变。


覆盖带50 μ 升1 × KHM吹打慢慢使用凝胶加载缓冲液尖端在顶部层。
注意:一个相间应1之间可以观察到× KHM和30%OptiPrep TM 在1 × KHM。


离心含有SA的超速离心管mple以100,000 ×g下在4℃下用于在加速设置(90分钟加速)3和减速在日立工机4 HIMAC 使用S55S-2080转子CS150NX微超速离心机(日立)。离心后,释放的囊泡将集中在1 × KHM中1x KHM和30%OptiPrep TM 之间的界面上。
收集囊泡通过取200 μ 升离心后的上清液从顶部。将上清液转移至厚壁聚碳酸酯管(Beckman Coulter,容量为0.5 ml,343776)中。添加300 μ 升1X KHM轻轻吹打缓冲到稀样品。
使用S120AT3-2061转子在Hitachi Koki himac CS150NX微型超速离心机(Hitachi)中以100,000 xg 在4°C下离心30分钟,以旋转囊泡。
 


免疫印迹
通过以下步骤执行标准的免疫印迹程序:


添加20 μ 升1 × SDS-PAGE蛋白质加样缓冲液在管的底部,涡流8分钟以溶解囊泡样缓冲液使用细胞破碎(否决精灵? )。
添加DM至20 微升裂解缓冲液(配方甲11 )和在冰上孵育30分钟。
在4°C下以14,000 xg离心5分钟,收集上清液。
在上清液中加入5×SDS-PAGE蛋白质上样缓冲液(配方A 4 ),使终浓度达到1×SDS-PAGE蛋白质上样缓冲液。
在55°C下孵育DM样品和高旋转沉淀(HSP)样品30分钟。
根据货物蛋白的大小,将DM样品和HSP样品上样到15个楔形孔中10-15%的凝胶上。
在恒定的20 mA下运行SDS-PAGE,直到染料在室温下用完凝胶(约70分钟)为止。
在4°C下以0.3 A恒定的速度将蛋白质转移到PVDF膜上1-2 小时。
在室温下,在PBST中用5%印迹级阻滞剂(配方A 7 )封闭PVDF膜30分钟。
在室温下将PVDF膜与一抗孵育1.5-2 h或在4°C过夜。
用PBST洗涤3×5分钟。
在室温下,将与HRP偶联的二抗孵育1小时。
用PBST洗涤3×5分钟。
与HRP底物ECL plus一起孵育,并在带有ImageLab 软件v4.0 的ChemiDoc TM 成像系统上成像。
 


数据分析


 


将ImageLab 软件v4.0中的免疫印迹图像导出为。tif 文件。
使用Photoshop和Adobe Illustrator ? 处理图像(图3 )。
 


D:\ Reformatting \ 2020-1-6 \ 1902842--1296郭玉松636915 \ Figs jpg \图3.jpg


图3. 从TGN将Frizzled6包装成囊泡的重构。用HA-Frizzled6转染COS7细胞。转染后第1天,使用表1中所示的指示剂进行TGN囊泡释放反应。浮选后的顶部馏分通过用指示的抗体进行免疫印迹分析。TGN46和Sec22分别用作监测TGN 出口和ER出口的阳性对照(Ma 等人,2018年重新发布)。    


菜谱


 


缓冲液
1×KHM(1公升)
将5ml的1M的Mg(OAC )2 ,20毫升的1M HEPES-KOH,pH 7.2的和110的1M 的KOAc 到0.5升的DDH 2 ?
调节至1 L
过滤缓冲区
储存在4°C
2×KHM(1公升)
加入10 mL的1M的Mg(OAC )2 ,将40ml 1M的HEPES-KOH,pH 7.2的和220的1M 的KOAc 到0.5升的DDH 2 ?
调节至1 L
过滤缓冲区
储存在4°C
10×KHM(1公升)
将50 ml 1 M Mg(OAc )2 和200 ml 1 M HEPES-KOH(pH 7.2)添加到0.5 L ddH 2 O
溶解108克KOAc
调节至1 L
过滤缓冲区
储存在4°C
5×SDS-PAGE蛋白加载缓冲液(50 ml)
在不断搅拌下,在装有12.5 ml 1 M Tris pH6.8的小烧杯中溶解3.75 g SDS,93.75 mg溴酚蓝
混合25毫升甘油
用ddH 2 O将体积调节至37.5 ml
等分试样至750 μ 升/管,并储存在-20℃下
加入250 μ 升之前使用βME到等分新鲜注:? 他在5×SDS-PAGE蛋白质加样缓冲液各试剂的最终浓度:7.5%SDS,50%甘油,250 毫摩尔Tris-HC 升和0.19%溴酚蓝, 25%βME(v / v)。
转移缓冲液(2 L)
溶解6.03克Tris和28.8克甘氨酸在1.5L的DDH 2 ? (?F 伊纳勒浓度:25 米的Tris ,192 mM的甘氨酸)
加入200毫升甲醇(?F 伊纳勒浓度:10%)
将ddH 2 O加到2 L
储存在4°C
SDS-PAGE运行缓冲液(1 L)
在900 ml ddH 2 O中添加100 ml 10 ×SDS-PAGE运行缓冲液


5%印迹级别的阻滞剂
在PBST中溶解5%印迹级别的阻滞剂(w / v)


PBS(1升)
在900 ml 10 ×PBS中加入100 ml 10 ×PBS


PBST
0.1%TWEEN ? 20(V / V)中的1×PBS


缓冲液E(1升)
一种。在800 ml ddH 2 O中加入50 ml pH 7.2的1 M HEPES-KOH,70 ml 1 M KOAc 和0.5 ml 1 M Mg(OAc )2       


b。在缓冲液中溶解45.54 g山梨糖醇和1.9 g EGTA钾      


C。调节至1 L       


d。过滤缓冲区      


e。分装成小体积,并在-80°C下储存       


F。在使用前添加1 × 浓度和5 mM DTT的蛋白酶抑制剂        


裂解缓冲液
添加0.5%的Triton ? X-100,1 × 蛋白酶抑制剂和1mM DTT中KHM缓冲


 


储备溶液
10×PBS,pH 7.4(2升)
将160 g NaCl ,4 g KCl ,28.8 g Na 2 HPO 4 和4.8 g KH 2 PO 4 溶于1.9 L ddH 2 O
用NaOH将pH调节至7.4
将ddH 2 O加到2 L
通过高压灭菌器灭菌并保存在RT中
10×SDS-PAGE运行缓冲液(1 L)
将30.3 g Tris,144.4 g甘氨酸和10 g SDS溶于1 L ddH 2 O
存放在RT
1 M KOAc (1升)
溶解98.14 g乙酸钾1 L ddH 2 O
通过高压灭菌器灭菌并保存在RT中
1 M HEPES-KOH,pH 7.2(1升)
将238.3 g HEPES溶于0.5 L ddH 2 O
用KOH调节pH到7.2
将ddH 2 O加到1 L
存放在RT
1 M镁(OAc )2 (1升)
将155.41 g Mg(OAc )2 溶于1 L ddH 2 O
通过高压灭菌器灭菌并保存在RT中
1.5 M Tris-HCl,pH 8.8(1升)
将181.7 g Tris溶于0.8 L ddH 2 O
用HCl调节pH值至8.8
将ddH 2 O加到1 L
通过高压灭菌器灭菌并保存在RT中
1 M Tris-HCl,pH 6.8(1 L)
将121.1 g Tris溶于0.8 L ddH 2 O
用HCl将pH调节至6.8
将ddH 2 O加到1 L
通过高压灭菌器灭菌并保存在RT中
10%SDS(100毫升)
将10克SDS溶于90毫升ddH 2 O中
存放在RT
10%APS(50毫升)
将5 g SDS溶解在50 ml ddH 2 O中
储存在4°C
3%NaN 3 (50毫升)
将1.5 g NaN 3 溶于50 ml ddH 2 O
存放在RT
40 mg / ml洋地黄皂苷(10 ml)
将400 mg洋地黄皂苷溶解在10 ml ddH 2 O中
等分试样的体积为30μl ,储存在-20°C
100×蛋白酶抑制剂(1毫升)
将2种蛋白酶抑制剂鸡尾酒片剂溶于1 ml ddH 2 O
等分试样的体积为30μl ,储存在-20°C
500 mM DTT(1毫升)
将0.077 g DL-二硫苏糖醇溶于1 ml ddH 2 O
等分试样的体积为30μl ,储存在-20°C
ATP再生系统(20毫升)
在20 ml 1 × KHM中溶解2.04 g磷酸肌酸,40 mg肌酸激酶,101.44 mg ATP
分装小份样品并储存在-80°C
10 mM GTP(20毫升)
将113.43 mg ATP溶于20 ml 1 × KHM中
分装小份样品并储存在-80°C
1毫克/毫升聚乙烯亚胺(100毫升)
将0.1 g 聚乙烯亚胺溶于100 ml ddH 2 O
通过过滤0.22 微米的过滤器
分装小份样品并储存在-80°C




致谢


 


该协议是根据先前报告中描述的先前工作进行修改的(Kim 等人,2005; Yuan 等人,2017 ; Ma 等人,2018; Niu 等人,2019)。这项工作得到了香港研究资助局的资助。YGYG的资助金26100315、16102218、16103319和16101116是HHMI在2009-2013年期间的博士后研究助理。


 


利益争夺


 


作者声明没有利益冲突或利益冲突。


 


参考文献


 


Guo,Y.,Sirkis ,DW和Schekman ,R.(2014年)。反式高尔基体网络上的蛋白质分选。Annu Rev Cell Dev Biol 30:169-206。
Kim,J.,Hamamoto ,S.,Ravazzola ,M.,Orci ,L。和Schekman ,R。(2005)。淀粉样蛋白前体蛋白和早老素1的解偶联包装到外壳蛋白复合物II囊泡中。生物化学杂志280(9):7758-7768。
Kim,J.,Kleizen ,B.,Choy,R.,Thinakaran ,G.,Sisodia ,SS和Schekman ,RW(2007)。分泌途径早期的γ-分泌酶的生物发生。J Cell Biol 179(5):951-963。
Ma,T.,Li,B.,Wang,R.,Lau,PK,Huang,Y,Jiang,L.,Schekman ,R.和Guo,Y.(2018)。反式高尔基体网络中平面细胞极性蛋白Frizzled6和Vangl2的差异分选机制。生物化学杂志293(22):8410-8427。
Merte ,J.,Jensen,D.,Wright,K.,Sarsfield,S.,Wang,Y.,Schekman ,R。和Ginty ,DD(2010)。Sec24b在神经管闭合期间选择性分选Vangl2以调节平面细胞极性。Nat Cell Biol 12(1):41-46; pp 41-48。
牛,L.,马,T.,杨,F.,闫,B.,唐X.,尹H.,吴Q.,黄Y.,姚,ZP,王J.,郭,Y.和Hu,J.(2019)。Atlastin介导的膜束缚对于货物移动和从内质网退出至关重要。PROC国家科科学院科学USA 116(28):14029-14038。
Ponnambalam,S.,Girotti ,M.,Yaspo ,ML,Owen,CE,Perry,AC,Suganuma ,T.,Nilsson,T.,Fried,M.,Banting,G.和Warren,G.(1996)。大鼠TGN38的灵长类同源物:一级结构,表达和功能含义。J Cell Sci 109(Pt 3):675-685。
Wakana ,Y.,van Galen,J.,Meissner,F.,Scarpa,M.,Polishchuk ,RS,Mann,M.和Malhotra,V.(2012)。新型运输工具,可将选择性货物从反高尔基网络运输到细胞表面。EMBO J 31(20):3976-3990。
Yuan,L.,Baba,S.,Bajaj,K. and Schekman ,R.(2017年)。无细胞的胶原蛋白包被的胶原蛋白I载体的产生。Bio - p rotoc ol 7(22):e2450 。
Yuan,L.,Kenny,SJ,Hemmati ,J.,Xu,K. and Schekman ,R.(2018年)。TANGO1和SEC12与前胶原I共同包装,以促进大型COPII载体的产生。PROC国家科科学院科学美国阿115(52):E12255-E12264。

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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tang, X., Yang, F. and Guo, Y. (2020). Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network. Bio-protocol 10(5): e3537. DOI: 10.21769/BioProtoc.3537.
  2. Ma, T., Li, B., Wang, R., Lau, P. K., Huang, Y., Jiang, L., Schekman, R. and Guo, Y. (2018). A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network. J Biol Chem 293(22): 8410-8427.
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