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Cell-free Generation of COPII-coated Procollagen I Carriers
COPII包被的前胶原I载体的无细胞生成   

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Abstract

The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the isolation and detection of reconstituted COPII-coated carriers. This cell-free reaction can be used to test conditions that stimulate or suppress the packaging of PC1 into COPII-coated carriers.

Keywords: COPII(COPII), Collagen(胶原), Membrane(膜), Budding(芽殖), Reconstitution(重建)

Background

The coat protein complex II (COPII) plays an essential role in transporting secretory cargos from the endoplasmic reticulum (ER) en route to the Golgi apparatus. The genes required for cargo traffic from the ER were discovered in genetic studies in yeast and the precise roles of the protein products of the genes required for vesicle budding were elucidated with the aid of a cell-free vesicle budding reaction supplemented with purified components (Novick et al., 1981; Kaiser et al., 1990; Barlowe et al., 1994). A similar reaction was developed to detect the role of COPII in cargo traffic from the ER in cultured mammalian cells (Kim et al., 2005). Mammalian COPII-coated vesicles are approximately 80-100 nm in diameter, which is seemingly too small to accommodate large secretory cargos such as the rigid 300 nm procollagen I (PC1) triple helical rod. Despite the potential size discrepancy, COPII is essential for the secretion of large cargos including PC1 (Boyadjiev et al., 2006). Recently, we reported the existence of bona fide large COPII-coated PC1 carriers, exceeding 300 nm in diameter, in cells evaluated by stochastic optical reconstruction microscopy (STORM), correlated light electron microscopy (CLEM) and live-cell imaging (Gorur et al., 2017). Cell-free COPII budding reactions that successfully reconstituted small COPII vesicles did not allow the detection of large COPII-coated PC1 carriers (Fromme and Schekman, 2005). Therefore, we devised an alternative vesicle budding protocol to allow the detection of PC1 packaged into large COPII vesicles as well as the characterization of both small and large COPII-coated vesicles. Using this new protocol, we showed that the capture of PC1 into large COPII vesicles requires COPII proteins and the GTPase activity of the COPII subunit SAR1 (Gorur et al., 2017).

Materials and Reagents

  1. Falcon® 150 mm TC-treated cell culture dish (Corning, Falcon®, catalog number: 353025 ) or equivalent
  2. BioExpress GeneMate 50 ml centrifuge tubes (BioExpress, Greiner Bio One, catalog number: C-3394-4 ) or equivalent
  3. BioExpress GeneMate racked pipet tips, low retention, 200 μl (BioExpress, catalog number: P-1234-200)
    Manufacturer: Biotix, catalog number: P-1234-200CS .
  4. BioExpress GeneMate racked pipet tips, low retention, 1,000 μl (BioExpress, catalog number: P-1234-1000)
    Manufacturer: Biotix, catalog number: P-1234-1000CS .
  5. BioExpress GeneMate 15 ml centrifuge tubes (BioExpress, Greiner Bio One, catalog number: C-3394-2 ) or equivalent
  6. Falcon® 100 mm TC-treated cell culture dish (Corning, Falcon®, catalog number: 353003 ) or equivalent
  7. Amicon® Ultra-15 ml centrifugal filter unit with Ultracel-3K membrane (Merck, catalog number: UFC900324 )
  8. Amicon® Ultra-0.5 ml centrifugal filter unit with Ultracel-3K membrane (Merck, catalog number: UFC500324 )
  9. Oxygen® 1.5 ml MAXYMmum recoveryTM microcentrifuge tube (low retention) (Corning, Axygen®, catalog number: MCT-150-L-C )
  10. Microscope slides (Fisher Scientific, catalog number: 12-550-343 ) or equivalent
  11. Microscope cover glass (Fisher Scientific, catalog number: 12-542A ) or equivalent
  12. Tube, 7 x 20 mm, thickwall, polycarbonate (Beckman Coulter, catalog number: 343775 )
  13. Prot/Elec tips (gel loading tips) (Bio-Rad Laboratories, catalog number: 2239915 )
  14. Cell scraper 25 cm (SARSTEDT, catalog number: 83.1830 ) or equivalent
  15. Corning 1 L filter system 0.22 μm (Corning, catalog number: 431098 ) or equivalent
  16. Steriflip® 50 ml filter 0.22 μm (Merck, catalog number: SCGP00525 ) or equivalent
  17. Posi-click 1.7 ml micro-centrifuge tube (Danville Scientific, catalog number: C2170 (1001002)) or equivalent
  18. Microfuge tube, polypropylene, 1.5 ml (Beckman Coulter, catalog number: 357448 )
  19. Cuvettes (SARSTEDT, catalog number: 67.742 )
  20. Immobilon®-P transfer membrane PVDF 0.45 μm (Merck, catalog number: IPVH00010 )
  21. HT-1080 human fibrosarcoma (ATCC, catalog number: CCL-121 ) for cytosol preparation
    Note: Other fast-growing cell lines that support PC1 secretion may also be used for this purpose.
  22. IMR-90 human lung fibroblasts (Coriell Cell Repositories at the National Institute on Aging, Coriell Institute for Medical Research) (Coriell Institute, catalog number: I90-83 ) for donor membrane preparation
    Note: Other cell lines that express endogenous PC1 and prolific at PC1 secretion may be used for this purpose. For this reaction, it is important to use young IMR-90 with cumulative Population Doubling Level (PDL) lower than 37.5, because aged cells secrete significantly less PC1. PDL was calculated using a standard formula: cumulative PDL = initial PDL + 3.32 [log (current cell yield) - log (cell plated)].
  23. Phosphate-buffered saline (PBS, pH 7.4)
  24. cOmpleteTM, EDTA-free, protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 05056489001 )
  25. Bio-beadsTM SM-2 adsorbent media (Bio-Rad Laboratories, catalog number: 1523920 )
  26. Bio-Rad protein assay dye reagent concentrate (Bradford) (Bio-Rad Laboratories, catalog number: 5000006 )
  27. Liquid nitrogen
  28. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  29. HyClone® trypan blue solution (GE Healthcare, HyCloneTM, catalog number: SV30084 )
  30. OptiPrepTM density gradient medium (Sigma-Aldrich, catalog number: D1556 )
  31. NovexTM WedgeWellTM 4-20% Tris-glycine gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: XP04205BOX )
  32. Antibodies
    1. Rabbit anti-PC1 (LF-41) was a gift from L. Fisher (National Institute of Dental and Craniofacial Research, Bethesda, MD), and it was used at 1:5,000
    2. Rabbit anti ribophorin I, ERGIC53, and SEC22B were made in-house and they were used at 1:5,000
    3. Mouse anti HSP47 (Enzo Life Sciences, catalog number: ADI-SPA-470-D ), and it was used at 1:5,000
  33. PierceTM ECL 2 Western blotting substrate (Thermo Fisher Scientific, catalog number: 32132 )
  34. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3294-100G )
  35. Life Science Seradigm premium grade fetal bovine serum (FBS) (VWR, catalog number: 1500-500 )
  36. DMEM, GlutaMAXTM (Thermo Fisher Scientific, GibcoTM, catalog number: 10566016 )
  37. HEPES (Sigma-Aldrich, catalog number: RDD002-1KG )
  38. Potassium hydroxide (KOH)
  39. D-Sorbitol (Sigma-Aldrich, catalog number: S1876-5KG )
  40. Potassium acetate (KoAc) (Fisher Scientific, catalog number: BP364-500 )
  41. Magnesium acetate tetrahydrate (MgoAc) (Sigma-Aldrich, catalog number: M0631-500G )
  42. Sodium dodecyl sulfate (SDS) (Avantor Performance Materials, J.T.Baker®, catalog number: 4095-02 )
  43. Glycerol (AMRESCO, catalog number: M152-4L )
  44. Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
  45. Glycine (Fisher Scientific, catalog number: BP381-5 )
  46. 2-Mercaptoethanol (βME) (AMRESCO, catalog number: M131-100ML )
  47. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
  48. Tris base (Fisher Scientific, catalog number: BP152-5 )
  49. Triton® X-100 (Sigma-Aldrich, catalog number: X100-500ML )
  50. TWEEN® 20 (Sigma-Aldrich, catalog number: P7949-500ML )
  51. Tris-buffered saline (TBS, pH 7.6)
  52. Digitonin (Sigma-Aldrich, catalog number: D141-500MG )
  53. Dimethyl sulfoxide (DSMO) (Sigma-Aldrich, catalog number: D8418-100ML )
  54. Trypsin inhibitor from glycine max (soybean) (Sigma-Aldrich, catalog number: T9003 )
  55. Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: 203637 )
  56. Creatine phosphate (Sigma-Aldrich, catalog number: 2380-25GM )
  57. Creatine kinase (Roche Diagnostics, catalog number: 10127566001 )
  58. Adenosine 5’-triphosphate (ATP) (GE Healthcare, catalog number: 27-1006-01 )
  59. GTP 100 mM Li Salt (Sigma-Aldrich, Roche Diagnostics, catalog number: 11140957001 )
  60. Methanol (Fisher Scientific, catalog number: A452-4 )
  61. Mammalian cell culture medium (see Recipes)
  62. Buffer solutions (see Recipes)
    1. B88
    2. B88-0
    3. Sample buffer (5x)
    4. Buffer C
    5. Sample buffer C (1x)
    6. Transfer buffer
    7. HK buffer
    8. TBST
  63. Stock solutions (see Recipes)
    1. 1 M HEPES pH 7.2
    2. 10% SDS
    3. Digitonin stock
    4. Trypsin inhibitor stock
    5. 0.5 M LiCl
    6. ATP regeneration system (ATP r.s.)
    7. GTP

Equipment

  1. SorvallTM ST16R centrifuge, TX-200 Swinging Bucket Rotor, 400 ml Round Buckets, 4 x 50 ml, 9 x 15 ml conical adapters (Thermo Fisher Scientific, model: SorvallTM ST 16R , catalog number: 75818382) or equivalent
  2. TLA-55 ultracentrifuge rotor (Beckman Coulter, model: TLA-55 , catalog number: 366725)
  3. Centrifuge 5430R, refrigerated with fixed angle rotor FA-45-30-11 (Eppendorf, model: 5430 R , catalog number: 5428000015)
  4. Light microscope with a 16x or 25x objective (any simple or compound light microscope is fine)
  5. S-24-11-AT swinging bucket rotor (Eppendorf, catalog number: 5409715003 )
  6. Spectronic Genesys 5 spectrophotometer (Spectronic Instruments) or equivalent
  7. Table top ultracentrifuge, we used Optima MAX-XP, Optima TL, and Optima TL-100 for this experiment (Beckman Coulter, models: OptimatTM MAX-XP , OptimaTM TL , OptimaTM TL-100 )
  8. Microman® positive-displacement pipet M250 (Gilson, catalog number: F148505 )
  9. Microman® capillary pistons for M250 (Gilson, catalog number: F148114 )
  10. TLS-55 swinging bucket ultracentrifuge rotor (Beckman Coulter, model: TLS-55 , catalog number: 346936)
  11. TLS-55 adapter, Delrin, for 7 x 20 mm tubes (Beckman Coulter, catalog number: 358615 )
  12. Micro tube mixer MT-360 (TOMY SEIKO, model: MT-360 )
  13. BioExpress GeneMate GyroMixer XL (BioExpress, GeneMate, catalog number: R-3200-1XL ) or equivalent platform rotator
  14. ChemiDocTM MP Imaging System (Bio-Rad Laboratories, model: ChemiDocTM MP )
  15. Small beaker

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 cultured HT-1080 cells (prepared ahead of time, will be used in the reaction, Figure 1)


    Figure 1. Schematic overview of the experimental procedure. The preparations of cytosol and donor membrane for the budding reaction were described in Procedure A and Procedure B, respectively. The assembly of budding reactions and isolation of vesicles from budding reactions were described in Procedure C. Packaging efficiency of COPII cargos were assessed by immunoblotting as described in Procedure D (Republished from Gorur et al., 2017 with modifications).

    1. Culture 20 x 15 cm plates of HT-1080 to 95% confluent in 30 ml culture medium per plate.
      Note: HT-1080 was chosen because it supports the secretion of overexpressed PC1, while it does not express PC1 endogenously and thus minimized background. Its fast growth rate also makes it easy to scale-up for cytosol preparation.
    2. Remove media and wash cells with 10 ml PBS/plate, repeat the wash one more time, and remove as much of PBS as possible.
    3. Scrape cells on ice and collect with 1 ml B88 buffer/plate (see Recipes) with protease inhibitors (used as suggested by manufacturer: 1 tablet per 50 ml).
      Note: This is done 5 plates at a time with 5 ml of B88 buffer. Collect cells from the first plate by resuspending with 5 ml of B88, and then use the same buffer to collect cells from the next 4 plates.
    4. Transfer cell suspension to a 50 ml Falcon tube.
    5. Add digitonin (40 mg/ml; see Recipes) to cell suspension to reach a final concentration of 80 μg/ml. Mix by inverting the tube a few times.
    6. Rotate for 30 min on a platform rotator at 4 °C.
    7. During the 30 min incubation, hydrate 4 g Bio-beadsTM (1 g per 5 x 15 cm plates) with 25 ml B88 buffer:
      1. Centrifuge at 300 x g for 5 min at Acceleration Setting (Accel) 9 (default) and Deceleration Setting (Decel) 7 using SorvallTM ST16R centrifuge.
      2. Discard supernatant and wash beads two more times with 25 ml B88 buffer.
      3. After the last wash, discard as much buffer as possible.
      Note: To remove buffer between Bio-beadsTM, press a P1000 tip to the bottom of the tube through beads then aspirate buffer with minimal disruption. This step should be immediately followed by the addition of crude cytosol to prevent Bio-beadsTM from drying out.
    8. After the 30 min digitonin incubation, centrifuge the cell suspension at 300 x g for 5 min.
    9. Take the supernatant (crude cytosol) and transfer to wash Bio-beadsTM.
    10. Incubate cytosol with Bio-beadsTM with mild agitation to absorb digitonin from the crude cytosol on a platform rotator at 4 °C overnight.
    11. The next morning, clarify the cytosol-beads mixture at 300 x g for 5 min at Decel 7.
    12. Recover supernatant and aliquot to about 14 x 1.5 ml polypropylene microfuge tubes.
    13. Centrifuge at 135,300 x g for 30 min in TLA-55 rotors at Accel 2 Decel 6.
    14. Collect supernatant conservatively and avoid disturbing sedimented material.
    15. Concentrate supernatant (cytosol) by centrifuging in 15 ml Amicon-3k concentrator at 4,000 x g for 4 x 10 min.
      Note: Cytosol was centrifuged 4 times for 10 min each time and mixed between each sedimentation to minimize protein precipitation.
    16. Further concentrate cytosol using 0.5 ml Amicon-3k concentrators at 14,000 x g for 3 x 10 min in a fixed angle rotor (FA-45-30-11, Eppendorf).
      Note: Cytosol was mixed between each sedimentation to minimize protein precipitation.
    17. Collect concentrated cytosol and measure protein concentration using Bradford reagents.
      Note: The concentration should be between 40-80 mg/ml.
    18. Freeze small aliquots (recommend 1.6 mg/aliquot) in liquid nitrogen and store at -80 °C for future use in budding reaction. Avoid repeated freeze-thaw cycles.

  2. Preparation of donor membrane (DM) from cultured human cells (prepare on the day of the reaction fresh, Figure 1)
    1. Culture 3 x 10 cm plates of IMR-90 to 95% confluent in 10 ml culture medium per plate.
      Note: Use young cells under PDL 37.5. Older cells are significantly less efficient at secreting PC1. IMR-90 was used to prepare donor membrane because it is the most efficient at PC1 secretion of all cell lines that we tested. Other cell lines such as sv-IMR90 and U-2OS may also be used to prepare donor membrane with lower budding efficiency (Gorur et al., 2017).
    2. Aspirate media from 3 x 10 cm plates of IMR-90.
    3. Wash each plate with 10 ml PBS.
    4. Add 0.5 ml 0.25% trypsin to each plate and incubate at RT for 5 min.
    5. Collect cells from each plate with 6 ml PBS buffer into 2 x 15 ml tubes.
    6. Add 25 μl 10 mg/ml trypsin inhibitor (see Recipes) to each tube and mix well.
    7. Centrifuge at 300 x g for 5 min.
    8. Discard supernatant and resuspend each cell pellet in 1 ml B88 buffer with low retention tips.
      Note: Dislodge the cell pellet by gently tapping it prior to the addition of buffer. Use low retention tips for all future steps.
    9. Add B88 buffer to each tube so that the final volume in each 15 ml tube is 6 ml.
    10. Add 3 μl 40 mg/ml digitonin to each tube so that final concentration is 20 μg/ml.
    11. Mix well and incubate on ice for 5 min.
    12. Add 8 ml B88 buffer to each tube and centrifuge at 300 x g for 5 min.
    13. Discard supernatant and resuspend each cell pellet in 1 ml B88 buffer and transfer to 2 x 1.5 ml low retention microcentrifuge tubes.
    14. Mix 3 μl of trypan blue and 3 μl of cells on a glass slide, carefully lay a cover slip over the sample, then check percentage of permeabilized cells under a light microscope with a 16x or 25x objective.
      Note: 100% of cells should be permeabilized at this stage. Blue nuclei and clear to light brown ER surrounding each blue nucleus should be observed.
    15. Centrifuge at 300 x g for 5 min in a swinging bucket rotor (S-24-11-AT).
      Note: Perform all subsequent centrifugations in a swinging bucket rotor for maximum recovery.
    16. Discard supernatant and resuspend each pellet in 1 ml B88 buffer containing 0.5 M LiCl (see Recipes) in B88.
    17. Incubate on ice for 5 min, then centrifuge at 300 x g for 5 min.
    18. Discard supernatant and resuspend each pellet in 1 ml B88 buffer.
      Note: Dislodge the cell pellet by gently tapping the tube prior to the addition of buffer.
    19. Incubate on ice for 5 min, then centrifuge at 300 x g for 5 min.
    20. Discard supernatant and resuspend each pellet in 1 ml B88-0 buffer (see Recipes).
    21. Centrifuge at 300 x g for 5 min.
    22. Discard supernatant and resuspend both pellets in 200 μl B88-0. This is a working stock of DM.
    23. Determine the concentration of DM in the working stock:
      1. Make a 1 to 50 dilution of DM in B88-0.
      2. Blank with B88-0 and measure the optical density of diluted DM sample at a wavelength of 600 nm (OD600) using a spectrophotometer. The reading is the ‘OD600’ of the diluted sample.
        Note: OD600 is used as a unit of concentration in this protocol.
      3. Calculate the concentration of the working stock by multiplying the OD600 of the diluted sample with 50.
        Note: If the OD600 of the working stock is above 2, then there will be enough of DM for 8 x 100 μl budding reactions with the final OD600 of 0.5.
    24. Calculate the volume of working stock to be added to each budding reaction using the equation:

      C1V1 = C2V2

      where, C is the concentration of DM in OD600 and V is the volume in μl.

  3. Reconstitution of COPII coated vesicles
    1. In low retention tubes, assemble budding reactions by adding ingredients from left to right in Table 1. Each 100 μl reaction contains ATP regeneration system (1 mM ATP, 40 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase), 3 mM GTP, 20 ng/μl SAR1B, 10 ng/μl SEC23A/24D, 10 ng/μl SEC13/31A, 2 μg/μl cytosol, and OD600 of 0.5 for DM.
      Note: Add B88-0, nucleotides (an ATP r.s. and GTP, see Recipes) and recombinant COPII proteins first. Mix well by pipetting and briefly centrifuge to collect liquid at the bottom of each tube. Then add DM and mix by pipetting up and down gently until homogenous. Add cytosol last and mix by gentle pipetting.

      Table 1. Sample calculation of cell-free PC1 budding reactions. Each row represents a single cell-free reaction. The total volume of each reaction is 100 μl. Each column represents a component of the reaction, and the volume added to the reaction in μl.


    2. Incubate reactions at 30 °C (or on ice as a negative control) for 1 h.
    3. Isolate COPII carriers of PC1 with the following centrifugation steps (Figure 1).
    4. Centrifuge at 7,000 x g for 10 min.
    5. Place 50 μl of 60% OptiPrepTM at the bottom of a 7 x 20 mm ultracentrifugation tube with positive-displacement pipet Microman® M250.
      Note: The OptiPrepTM gradient purchased from Sigma-Aldrich is a 60% (w/v) solution.
    6. Recover 85 μl 7,000 x g supernatant and mix with the 60% OptiPrepTM in a 7 x 20 mm ultracentrifugation tube until homogenous, resulting in a 22.2% (w/v) OptiPrepTM mixture.
      Note: Avoid introducing bubbles.
    7. Overlay with 100 μl 18% (w/v) OptiPrepTM in B88 by slowly pipetting against the wall using a gel-loading tip.
      Important Note: An interphase should be observed between 18% OptiPrepTM and the 22.2% 7,000 x g supernatant OptiPrepTM mixture at this stage.
    8. Overlay with 10 μl B88 by pipetting slowly using a gel-loading tip.
      Important Note: An interphase should be observed between B88 and 18% OptiPrepTM in B88.
    9. Centrifuge at 55,000 rpm (or 258,488 x g) for 90 min at the Acceleration Setting (Accel) 2 and Decel 6 in TLS-55 rotors and adaptors for 7 x 20 mm tubes.
    10. Collect lipid vesicles from the top of the flotation gradient immediately after the centrifugation is over.

  4. Immunoblot (Western blot)
    Perform standard immunoblotting procedure by following the notes below:
    1. Add DM to 20 μl 1x sample buffer C (see Recipes) to reach a final concentration of 0.1 OD600/μl DM, then mix vigorously using a micro-tube mixer at max speed for 10 min at RT.
    2. Add 5x sample buffer (see Recipes) to float fractions.
    3. Incubate both DM sample and floated samples at 65 °C for 10 min.
    4. Load the desired amount of DM sample and the entire floated fractions onto a 15 wedged well 4-20% gradient gel.
    5. Run sample at constant 25 mA until dye runs out of the gel (about 75 min) at RT.
    6. Transfer protein onto a PVDF membrane at constant 0.15 A for 16 h at 4 °C.
      Important Note: The transfer condition was optimized for PC1.Conditions may vary for other proteins of interest.
    7. Block with 5% non-fat milk (w/v) in TBST (see Recipes) for 30 min at RT.
    8. Incubate with primary antibodies at RT for 2.5 h.
      Important Note: The temperature and duration were optimized for the rabbit anti PC1 antibody LF-41, which was used at 1:5,000 in 1% BSA (w/v) TBST. Each aliquot can be stored at 4 °C and reused up to 7 times without compromising the detection. An ER marker (ribophorin I) is included as a negative control, whereas regular COPII cargos (ERGIC53 and SEC22B) are used as positive controls.
    9. Wash with TBST 3 x 5 min.
    10. Incubate with secondary antibodies conjugated with HRP at RT for 1 h.
    11. Wash with TBST 3 x 5 min.
    12. Develop 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 ImageJ to process immunoblot images (rotating, cropping, adjusting brightness and contrast when necessary).
  3. Prepare figures with Adobe Illustrator® or equivalent software (Figure 2).


    Figure 2. COPII is required to package PC1 into reconstituted vesicles. Budding requirements of PC1 and HSP47 (a collagen-specific chaperone) were assessed under different incubation conditions described in Table 1. The top fraction after flotation was taken from each sample and analyzed by immunoblotting. Ribophorin I is an ER resident protein that serves as a negative control. ERGIC53 and SEC22B are found in conventional COPII vesicles and serve as positive controls. (Republished from Gorur et al., 2017)

Recipes

  1. Mammalian cell culture medium
    Add 10% FBS to DMEM

  2. Buffer solutions
    1. B88 (1 L)
      1. Add 20 ml 1 M HEPES pH 7.2 buffer to 0.5 L ddH2O
      2. Dissolve 45.54 g sorbitol, 14.72 g KoAc, and 1 g MgoAc
      3. Adjust volume to 1 L
      4. Filter the buffer
      5. Store at 4 °C
    2. B88-0 (50 ml)
      1. Add 1 ml 1 M HEPES pH 7.2 buffer to 30 ml ddH2O
      2. Dissolve 2.28 g sorbitol and 0.7 g KoAc
      3. Adjust volume to 50 ml
      4. Filter the buffer
      5. Store at 4 °C
    3. 5x sample buffer (10 ml)
      1. Dissolve 1 g of SDS, 5 mg bromophenol blue in a small beaker with 2 ml 1 M Tris pH 6.8 with constant stirring
      2. Mix in 3 ml glycerol
      3. Adjust volume to 10 ml with ddH2O
      4. Aliquot to 200 μl/tube and store at -20 °C
      5. Add 10 μl βME to an aliquot fresh before use
    4. Buffer C (1 ml)
      1. Add 10 μl of 1 M Tris pH 7.6, 20 μl of 5 M NaCl, 100 μl of Triton® X-100, and 100 μl 10% SDS
      2. Add water to 1 ml, mix well and store at 4 °C
    5. Sample buffer C (1x)
      Dilute 5x sample buffer to 1x with buffer C
    6. Transfer buffer
      1. Dissolve 6.06 g Tris and 28.8 g glycine in 1.5 L water
      2. Add 1 ml 10% SDS
      3. Add ddH2O to 2 L
      4. Store at 4 °C, use within 2 weeks
      Note: For best and consistent result, do not reuse this transfer buffer.
    7. HK buffer
      1. Dissolve 785.2 mg of KoAc in 30 ml ddH2O
      2. Add 1 ml of HEPES, pH 7.2
      3. Adjust volume to 50 ml and store at 4 °C
    8. TBST
      0.1% TWEEN® 20 (v/v) in 1x TBS

  3. Stock solutions
    1. 1 M HEPES pH 7.2
      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
    2. 10% SDS
      Dissolve 1 g of SDS in 9 ml of ddH2O
      Store at RT
    3. Digitonin stock
      Dissolve 40 mg digitonin in 1 ml DMSO
      Save small aliquots at -20 °C
    4. Trypsin inhibitor stock
      Dissolve 10 mg trypsin inhibitor in 1 ml B88
      Save small aliquots at -20 °C
      Avoid repeated freeze-thaws
    5. 0.5 M LiCl
      Dissolve 212 mg LiCl in 10 ml B88
      Store at 4 °C
    6. ATP regeneration system (ATP r.s.)
      Dissolve 2.04 g creatine phosphate, 40 mg creatine kinase, 101.44 mg ATP in 20 ml B88
      Store small aliquots at -80 °C
    7. GTP
      Dilute 400 μl of 100 mM GTP with 3.6 ml HK buffer
      Store small aliquots at -80 °C

Acknowledgments

We thank the staff at the University of California Berkeley Cell Culture Facility Ann Fisher and Alison Kililea. We also thank past and present members of the Schekman Lab, in particular Liang Ge and Yusong Guo for helpful discussions on optimizing this protocol. RS is supported as an Investigator of the Howard Hughes Medical Institute and the UC Berkeley Miller Institute of Science. LY was supported in part by the Tang family fellowship. This protocol was modified from previous work described in Kim et al., 2005. The authors declare no conflict of interest or competing interests.

References

  1. Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M. and Schekman, R. (1994). COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77(6): 895-907.
  2. Boyadjiev, S. A., Fromme, J. C., Ben, J., Chong, S. S., Nauta, C., Hur, D. J., Zhang, G., Hamamoto, S., Schekman, R., Ravazzola, M., Orci, L. and Eyaid, W. (2006). Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking. Nat Genet 38(10): 1192-1197.
  3. Fromme, J. C. and Schekman, R. (2005). COPII-coated vesicles: flexible enough for large cargo? Curr Opin Cell Biol 17(4): 345-352.
  4. Gorur, A., Yuan, L., Kenny, S. J., Baba, S., Xu, K. and Schekman, R. (2017). COPII-coated membranes function as transport carriers of intracellular procollagen I. J Cell Biol 216(6): 1745-1759.
  5. Kaiser, C. A. and Schekman, R. (1990). Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell 61(4): 723-733.
  6. 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.
  7. Novick, P., Ferro, S. and Schekman, R. (1981). Order of events in the yeast secretory pathway. Cell 25(2): 461-469.

简介

该协议的目的是在无细胞反应中产生COPII包被的前胶原I(PC1)载体。 COPII包被的PC1载体由供体膜,胞质溶胶,纯化的重组COPII蛋白和核苷酸重构。 该方案描述了供体膜和细胞溶胶的制备,反应的组装,以及复制的COPII包被的载体的分离和检测。 该无细胞反应可用于测试刺激或抑制PC1包装成COPII包被的载体的条件。
【背景】外壳蛋白复合体II(COPII)在从内质网(ER)途径到高尔基体的运输中起着至关重要的作用。来自ER的货物运输所需的基因在酵母的基因研究中被发现,并且借助于添加有纯化组分的无细胞囊泡萌芽反应来阐明囊泡出芽所需基因的蛋白质产物的精确作用(Novick 1981; Kaiser等人,1990; Barlowe等人,1994)。开发了类似的反应来检测COPII在培养的哺乳动物细胞中来自ER的货物运输中的作用(Kim等人,2005)。哺乳动物COPII包被的囊泡直径大约为80-100nm,似乎太小以至于不能容纳诸如刚性的300nm前胶原I(PC1)三股螺旋杆的大分泌性货物。尽管可能的尺寸差异,COPII对于包括PC1在内的大型货物的分泌是必不可少的(Boyadjiev等人,2006)。最近,我们报道了通过随机光学重建显微镜(STORM),相关光电子显微镜(CLEM)和活细胞成像(Gorur em)评估的存在真正大的COPII涂覆的PC1载体(直径超过300nm) 等。,2017)。成功地重建小COPII囊泡的无细胞COPII出芽反应不允许检测大COPII包被的PC1载体(Fromme和Schekman,2005)。因此,我们设计了一种替代的囊泡萌芽协议,以允许检测包装成大的COPII囊泡的PC1以及小和大的COPII包被的囊泡的表征。使用这种新的方案,我们表明将PC1捕获到大的COPII囊泡中需要COPII蛋白和COPII亚基SAR1的GTP酶活性(Gorur等人,2017)。

关键字:COPII, 胶原, 膜, 芽殖, 重建

材料和试剂

  1. Falcon 150mmTC-处理的细胞培养皿(Corning,Falcon?,目录号:353025)或等同物。
  2. BioExpress GeneMate 50ml离心管(BioExpress,Greiner Bio One,目录号:C-3394-4)或同等产品
  3. BioExpress GeneMate架式移液枪头,低保留,200μl(BioExpress,目录号:P-1234-200)
    制造商:Biotix,目录号:P-1234-200CS。
  4. BioExpress GeneMate架式移液枪头,低保留,1,000μl(BioExpress,目录号:P-1234-1000)
    制造商:Biotix,目录号:P-1234-1000CS。
  5. BioExpress GeneMate 15 ml离心管(BioExpress,Greiner Bio One,目录号:C-3394-2)或同等产品
  6. Falcon 100mmTC-处理的细胞培养皿(Corning,Falcon,目录号353003)或等同物。
  7. Amicon Ultra Ultra-15 ml Ultracel-3K离心过滤装置(Merck,目录号:UFC900324)
  8. Amicon Ultra Ultra-0.5ml带Ultracel-3K膜的离心过滤装置(Merck,目录号:UFC500324)
  9. 1.5毫升MAXYMmum回收TM离心管(低保留)(Corning,Axygen,目录号:MCT-150-LC) br />
  10. 显微镜载玻片(Fisher Scientific,产品目录号:12-550-343)或相当的
  11. 显微镜保护玻璃(Fisher Scientific,产品目录号:12-542A)或等同物
  12. 管,7×20毫米,厚壁,聚碳酸酯(贝克曼库尔特,目录号:343775)
  13. Prot / Elec提示(凝胶加样提示)(Bio-Rad Laboratories,目录号:2239915)
  14. 细胞刮刀25厘米(SARSTEDT,目录号:83.1830)或同等产品
  15. Corning 1 L过滤系统0.22μm(Corning,目录编号:431098)或同等产品
  16. Steriflip 50毫升过滤器0.22微米(Merck,产品目录号:SCGP00525)或同等产品
  17. Posi-click 1.7ml微型离心管(Danville Scientific,目录号:C2170(1001002))或同等产品
  18. 微量离心管,聚丙烯,1.5毫升(Beckman Coulter,目录号:357448)
  19. 比色杯(SARSTEDT,目录号:67.742)
  20. Immobilon®-P转移膜PVDF 0.45μm(Merck,目录号:IPVH00010)
  21. HT-1080人纤维肉瘤(ATCC,目录号:CCL-121)用于胞质溶胶制备
    注:其他支持PC1分泌的快速生长细胞系也可用于此目的。
  22. 供体膜制备的IMR-90人肺成纤维细胞(Coriell Institute for Ageing,Coriell Institute for Medical Research)(Coriell Institute,目录号:I90-83) 注意:表达内源性PC1并在PC1分泌中多产的其他细胞系可用于此目的。对于这种反应,使用年轻的IMR-90(累积种群倍增水平(PDL)低于37.5)是重要的,因为老化的细胞分泌显着更少的PC1。使用标准公式计算PDL:累积PDL =初始PDL + 3.32 [log(当前细胞产量) - log(细胞培养)]。
  23. 磷酸缓冲盐水(PBS,pH7.4)
  24. 完全不含EDTA的蛋白酶抑制剂混合物片剂(Roche Diagnostics,目录号:05056489001)
  25. Bio-beads TM SM-2吸附介质(Bio-Rad Laboratories,目录号:1523920)
  26. Bio-Rad蛋白测定染料试剂浓缩物(Bradford)(Bio-Rad Laboratories,目录号:5000006)
  27. 液氮
  28. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  29. HyClone台盼蓝溶液(GE Healthcare,HyClone TM,产品目录号:SV30084)
  30. OptiPrep TM密度梯度培养基(Sigma-Aldrich,目录号:D1556)
  31. Novex TM WedgeWell TM 4-20%Tris-甘氨酸凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:XP04205BOX)
  32. 抗体
    1. 兔抗PC1(LF-41)是由L.Fisher(National Institute of Dental and Craniofacial Research,Bethesda,MD)提供的一种礼物,其用量为1:5,000。
    2. 兔抗反式核糖核蛋白I,ERGIC53和SEC22B是内部制造的,它们以1:5,000的比例使用。
    3. 小鼠抗HSP47(Enzo Life Sciences,产品目录号:ADI-SPA-470-D),并以1:5,000使用。
  33. Pierce TM ECL 2 Western印迹底物(Thermo Fisher Scientific,目录号:32132)
  34. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3294-100G)
  35. 生命科学Seradigm特级胎牛血清(FBS)(VWR,目录号:1500-500)
  36. DMEM,GlutaMAX TM(Thermo Fisher Scientific,Gibco TM,产品目录号:10566016)
  37. HEPES(Sigma-Aldrich,目录号:RDD002-1KG)
  38. 氢氧化钾(KOH)
  39. D-山梨醇(Sigma-Aldrich,目录号:S1876-5KG)
  40. 乙酸钾(KoAc)(Fisher Scientific,目录号:BP364-500)
  41. 醋酸镁四水合物(MgoAc)(Sigma-Aldrich,目录号:M0631-500G)
  42. 十二烷基硫酸钠(SDS)(Avantor Performance Materials,J.T.Baker,目录号:4095-02)
  43. 甘油(AMRESCO,目录号:M152-4L)
  44. 溴酚蓝(Bio-Rad Laboratories,目录号:1610404)
  45. 甘氨酸(Fisher Scientific,目录号:BP381-5)
  46. 2-巯基乙醇(βME)(AMRESCO,目录号:M131-100ML)
  47. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-3)
  48. Tris碱(Fisher Scientific,目录号:BP152-5)
  49. Triton X-100(Sigma-Aldrich,目录号:X100-500ML)
  50. TWEEN 20(Sigma-Aldrich,目录号:P7949-500ML)
  51. Tris缓冲盐水(TBS,pH7.6)
  52. Digitonin(Sigma-Aldrich,目录号:D141-500MG)
  53. 二甲基亚砜(DSMO)(Sigma-Aldrich,目录号:D8418-100ML)
  54. 来自甘氨酸max(大豆)的胰蛋白酶抑制剂(Sigma-Aldrich,目录号:T9003)
  55. 氯化锂(LiCl)(Sigma-Aldrich,目录号:203637)
  56. 磷酸肌酸(Sigma-Aldrich,目录号:2380-25GM)
  57. 肌酸激酶(Roche Diagnostics,目录号:10127566001)
  58. 5'-三磷酸腺苷(ATP)(GE Healthcare,目录号:27-1006-01)
  59. GTP 100mM Li盐(Sigma-Aldrich,Roche Diagnostics,目录号:11140957001)
  60. 甲醇(Fisher Scientific,目录号:A452-4)
  61. 哺乳动物细胞培养基(见食谱)
  62. 缓冲溶液(见食谱)
    1. B88
    2. B88-0
    3. 样本缓冲区(5x)
    4. 缓冲区C
    5. 样本缓冲区C(1x)
    6. 传输缓冲区
    7. HK缓冲区
    8. TBST
  63. 库存解决方案(请参阅食谱)
    1. 1 M HEPES pH 7.2
    2. 10%SDS
    3. Digitonin股票
    4. 胰蛋白酶抑制剂股票
    5. 0.5 M LiCl
    6. ATP再生系统(ATP r.s.)
    7. GTP

设备

  1. Sorvall TM ST16R离心机,TX-200摆动转子,400ml圆桶,4×50ml,9×15ml锥形适配器(Thermo Fisher Scientific,型号:Sorvall TM TM > ST 16R,产品目录号:75818382)或同等产品
  2. TLA-55超速离心机转子(Beckman Coulter,型号:TLA-55,目录号:366725)
  3. 用定角转子FA-45-30-11冷冻离心机5430R(Eppendorf,型号:5430R,目录号:5428000015)
  4. 光学显微镜的16倍或25倍的目标(任何简单或复合光学显微镜都很好)
  5. S-24-11-AT摆动斗式转子(Eppendorf,货号:5409715003)
  6. Spectronic Genesys 5分光光度计(Spectronic Instruments)或同等产品
  7. 台式超速离心机中,我们使用Optima MAX-XP,Optima TL和Optima TL-100用于该实验(Beckman Coulter,型号:Optimat TM MAX-XP,Optima TM TM) TL,Optima TM TL-100)
  8. Microman正位移移液管M250(Gilson,目录号:F148505)
  9. 用于M250的Microman™毛细管活塞(Gilson,目录号:F148114)
  10. TLS-55摆动桶式超速离心机转子(Beckman Coulter,型号:TLS-55,目录号:346936)
  11. TLS-55适配器,Delrin,用于7 x 20 mm管(Beckman Coulter,目录号:358615)
  12. 微管混合器MT-360(TOMY SEIKO,型号:MT-360)
  13. BioExpress GeneMate GyroMixer XL(BioExpress,GeneMate,产品目录号:R-3200-1XL)或同等平台旋转器
  14. ChemiDoc TM成像系统(Bio-Rad Laboratories,型号:ChemiDoc TM TM)
  15. 小烧杯

软件

  1. ImageLab软件v4.0
  2. ImageJ

程序

注意:除非另有说明,否则所有操作均在冰上进行,所有离心操作均在4°C进行。

  1. 从培养的HT-1080细胞制备胞质溶胶(提前准备,将用于反应,图1)


    图1.实验程序的示意性概述分别在程序A和程序B中描述胞质溶胶和供体膜用于出芽反应的制备。在程序C中描述了出芽反应的组装和囊泡从出芽反应中的分离.COPII货物的包装效率通过如程序D(由Gorur等人于2017年重新公布)中所述的免疫印迹来评估, )。

    1. 培养20×15厘米的HT-1080到95%汇合在30毫升培养基每板平板。
      注意:选择HT-1080是因为它支持过度表达的PC1的分泌,而不是内源地表达PC1,因此使背景最小化。它的快速增长速度也使得放大制备胞质溶胶变得容易。
    2. 取出培养基并用10 ml PBS /平板清洗细胞,重复洗涤一次,尽可能多地除去PBS。
    3. 在冰上刮细胞并用蛋白酶抑制剂(如制造商建议:每50ml 1片)用1ml B88缓冲液/平板(参见食谱)收集。
      注意:这是用5毫升的B88缓冲液一次完成5个板。通过用5ml B88重悬细胞从第一块板收集细胞,然后使用相同的缓冲液收集接下来4块板的细胞。
    4. 将细胞悬液转移到50ml Falcon管中。
    5. 加入毛地黄皂苷(40毫克/毫升;见食谱)细胞悬液达到80微克/毫升的终浓度。
      倒转管子混合几次

    6. 在4°C的平台旋转器上旋转30分钟
    7. 在孵育30分钟期间,用25ml B88缓冲液水合4g Bio-beads TM(1g / 5×15cm平板)
      1. 使用Sorvall TM ST16R离心机,在加速设置(加速)9(默认)和减速设置(减速)7下,以300gxg离心5分钟。
      2. 丢弃上清,再用25毫升B88缓冲液洗两次珠。
      3. 最后一次洗涤后,尽可能多地放弃缓冲液。
      注意:要去除Bio-beads之间的缓冲液,请按P1000尖端到达试管底部,然后吸取缓冲液以最小的中断。这个步骤之后应该立即加入粗胞质以防止Bio-beads TM干燥。 >
    8. 孵育30分钟后,将细胞悬液以300×g离心5分钟。
    9. 取上清液(粗胞质溶胶),转移到Bio-beads TM 上洗。
    10. 使用Bio-beads TM轻微搅拌孵育胞质溶胶以在4℃的平台旋转器上从粗胞质溶胶中吸收毛地黄皂苷过夜。
    11. 第二天早上,在Decel7将300μgxg的细胞溶胶珠混合物澄清5分钟。
    12. 回收上清液,并分装到约14×1.5毫升聚丙烯microfuge管。
    13. 在Accel 2 Decel 6在TLA-55转子中在135,300×gg下离心30分钟。
    14. 保守收集上清液,避免干扰沉淀物。
    15. 在15ml Amicon-3k浓缩器中以4,000×gg离心浓缩上清液(胞质溶胶)4×10分钟。
      注意:Cytosol每次离心4次,每次10分钟,在每次沉淀之间混合,使蛋白质沉淀最小化。
    16. 使用0.5ml Amicon-3k浓缩器,在固定角转子(FA-45-30-11,Eppendorf)中以14,000×gg进一步浓缩细胞溶质3×10分钟。
      注意:细胞溶胶在每次沉淀之间混合,以尽量减少蛋白质沉淀。
    17. 收集浓缩的胞质溶胶并使用Bradford试剂测量蛋白质浓度。
      注意:浓度应该在40-80毫克/毫升之间。
    18. 在液氮中冷冻小份(建议1.6毫克/等分),储存在-80°C,以备将来用于出芽反应。避免反复冻融。

  2. 从培养的人细胞制备供体膜(DM)(在新鲜反应当天制备,图1)
    1. 培养3×10厘米的IMR-90到95%汇合在10毫升培养基每盘平板。
      注:使用PDL 37.5下的年轻细胞。老年细胞分泌PC1的效率明显降低。 IMR-90用于制备供体膜,因为它是我们测试的所有细胞系中PC1分泌最有效的。其他细胞系如sv-IMR90和U-20S也可用于制备芽生效率较低的供体膜(Gorur等,2017)。
    2. 从IMR-90的3×10厘米的平板上吸取培养基。
    3. 用10毫升的PBS洗每个板。

    4. 加入0.5 ml 0.25%胰蛋白酶到每个板上,并在室温孵育5分钟

    5. 用6毫升PBS缓冲液将每个平板上的细胞收集到2×15毫升的管中

    6. 加入25μl10 mg / ml胰蛋白酶抑制剂(见食谱),并充分混合

    7. 在300×g离心5分钟
    8. 丢弃上清,并重悬在1毫升B88缓冲液与低保留技巧每个细胞沉淀。
      注意:在加入缓冲液之前轻轻敲击细胞沉淀物。









    9. 将B88缓冲液加到每个试管中,使每个15ml试管的最终体积为6ml。

    10. 加入3μl40 mg / ml毛地黄皂苷至终浓度为20μg/ ml
    11. 充分混合,在冰上孵育5分钟。
    12. 加入8毫升B88缓冲液到每个管中,并在300×g下离心5分钟。
    13. 弃上清,重悬在1毫升B88缓冲液中的每个细胞沉淀,并转移到2×1.5毫升低保留微量离心管。
    14. 混合台盼蓝的3微升和细胞的3微升在载玻片上,小心地放置在样品上盖玻片,然后检查用16倍或25倍物镜在光学显微镜下透化细胞的百分比。
      注意:在这个阶段,100%的细胞应该是透化的。应该观察到蓝色的细胞核,并清楚地看到围绕着每个蓝色核的浅棕色ER。
    15. 在摆动斗式转子(S-24-11-AT)中以300gxg离心5分钟。
      注意:为了最大限度地恢复,在一个摆动的转子中进行所有后续的离心。
    16. 弃去上清液并将每个沉淀物重悬于含有0.5M LiCl的1ml B88缓冲液(见食谱)中B88。
    17. 在冰上孵育5分钟,然后在300×g离心5分钟。
    18. 弃去上清液,并将每个沉淀物重悬于1ml B88缓冲液中。
      注意:在加入缓冲液之前轻轻敲击试管,取出细胞沉淀。
    19. 在冰上孵育5分钟,然后在300×g离心5分钟。
    20. 丢弃上清,并重悬在1毫升B88-0缓冲区(见食谱)的每个颗粒。

    21. 在300×g离心5分钟
    22. 弃去上清液,并将两种沉淀物重悬于200μlB88-0中。这是DM的工作库存。
    23. 确定工作原料中DM的浓度:

      1. 在B88-0中稀释1到50倍的DM
      2. 用B88-0空白并使用分光光度计测量在600nm波长(OD 600)下稀释的DM样品的光密度。读数是稀释样品的“OD 600”。
      3. 通过将稀释的样品的OD 600乘以50来计算工作物质的浓度。
        注意:如果工作库存的OD 600 大于2,那么将会有足够的DM用于8 x 100μl萌芽反应与最终OD 600 0.5。
    24. 使用以下公式计算每个出芽反应中添加的工作原料量:

      C1V1 = C2V2

      其中C是OD 600的DM浓度,V是以μl计的体积。

  3. COPII包被的囊泡的重建
    1. 在低保留管中,通过在表1中从左至右添加成分来组装萌芽反应。每个100μl反应物含有ATP再生系统(1mM ATP,40mM磷酸肌酸,0.2mg / ml肌酸磷酸激酶),3mM GTP,20 ng /μl的SAR1B,10ng /μl的SEC23A / 24D,10ng /μl的SEC13 / 31A,2μg/μl的胞质溶胶,以及0.5的OD为600。
      注意:首先添加B88-0核苷酸(ATP r.s.和GTP,参见食谱)和重组COPII蛋白质。通过移液充分混合并短暂离心以收集每个管底部的液体。然后加入DM,轻轻地上下吸取混匀,直至均匀。最后加入细胞质液,并轻轻移液。

      表1.无细胞PC1出芽反应的样品计算。每行代表一个无细胞反应。每个反应的总体积是100μl。每列代表反应的一个组成部分,加入到反应中的体积(μl)。



    2. 在30°C孵育反应(或在冰上作为阴性对照)1小时。
    3. 用下面的离心步骤(图1)分离PC1的COPII载体。

    4. 7,000×g离心10分钟
    5. 在带有容积式移液管Microman M250的7×20mm超速离心管的底部放置50μl60%OptiPrep TM。
      注意:从Sigma-Aldrich购买的OptiPrep梯度梯度为60%(w / v)。
    6. 回收85μl7,000xg上清液,并在7×20mm超速离心管中与60%OptiPrep TM混合直至均匀,得到22.2%(w / v)OptiPrep 混合物。
      注意:避免引入气泡。
    7. 在B88中用100μl18%(w / v)的OptiPrepTM TM叠加,使用凝胶加样尖缓慢地吸干壁。
      与22.2%7,000 xg上清OptiPrep之间应该观察到相间 TM 混合物。

    8. 使用凝胶加样尖缓慢吸取10μlB88 重要提示:在B88中,B88和18%OptiPrep之间应该有一个相间的相位{TM} 。
    9. 在TLS-55转子的加速设定(加速)2和减速6以及适用于7×20毫米的管子的情况下以55,000rpm(或258,488×gg)离心90分钟。

    10. 在离心结束后立即收集浮选梯度顶部的脂质囊泡。

  4. 免疫印迹(蛋白质印迹)
    按照以下注意事项执行标准的免疫印迹程序:
    1. 将DM加入到20μl1x样品缓冲液C(参见配方)中以达到0.1 OD 600 /μlDM的终浓度,然后在室温下使用微管混合器以最大速度剧烈混合10分钟。
    2. 添加5倍的样品缓冲液(见食谱)以浮选分数。

    3. 在65°C温育DM样品和浮选样品10分钟
    4. 将所需量的DM样品和整个漂浮的部分加到15-楔形孔4-20%梯度凝胶上。
    5. 在恒定的25mA下运行样品,直到在室温下染料流出凝胶(约75分钟)。
    6. 将蛋白质在恒定的0.15A下在4℃下转移至PVDF膜上16h。
      重要提示:传输条件针对PC1进行了优化。对于其他感兴趣的蛋白质,条件可能会有所不同。
    7. 在室温下用TBST中的5%无脂牛奶(w / v)封闭30分钟(见食谱)。
    8. 在RT下孵育一次抗体2.5小时。
      重要注意事项:温度和持续时间针对在1%BSA(w / v)TBST中以1:5,000使用的兔抗PC1抗体LF-41进行优化。每个等分试样可以保存在4°C,重复使用7次而不影响检测。包括ER标记(核糖体蛋白I)作为阴性对照,而常规COPII货物(ERGIC53和SEC22B)用作阳性对照。
    9. 用TBST洗3次5分钟。

    10. 用与HRP结合的二抗在RT孵育1小时
    11. 用TBST洗3次5分钟。
    12. 使用ImageLab软件v4.0在HRP底物ECL plus和ChemiDoc TM成像系统上成像。

数据分析


  1. 将ImageLab软件v4.0中的免疫印迹图像导出为.tif文件
  2. 使用ImageJ处理免疫印迹图像(旋转,裁剪,必要时调整亮度和对比度)。
  3. 使用Adobe Illustrator ®或同等软件准备数字(图2)。


    图2. COPII需要将PC1包装到重组囊泡中。在表1中描述的不同温育条件下评估PC1和HSP47(胶原特异性伴侣)的萌芽要求。从每个样品取得浮选后的顶部馏分并通过免疫印迹进行分析。核糖核酸蛋白原I是一种ER驻留蛋白,可作为阴性对照。 ERGIC53和SEC22B存在于常规COPII囊泡中,并作为阳性对照。 (由Gorur等人,2017年重新出版)

食谱

  1. 哺乳动物细胞培养基
    添加10%FBS到DMEM

  2. 缓冲溶液
    1. B88(1 L)
      1. 加入20ml 1M HEPES pH7.2缓冲液至0.5L ddH2O 2
      2. 溶解45.54克山梨醇,14.72克KoAc和1克MgoAc
      3. 调整音量到1升
      4. 过滤缓冲区
      5. 在4°C储存
    2. B88-0(50毫升)
      1. 加入1ml 1M HEPES pH 7.2缓冲液至30ml ddH 2 O中
      2. 溶解2.28克山梨糖醇和0.7克KoAc
      3. 调整音量到50毫升
      4. 过滤缓冲区
      5. 在4°C储存
    3. 5倍样品缓冲液(10毫升)
      1. 溶解1克SDS,5毫克溴酚蓝在2毫升1M的pH值为6.8的小型烧杯中恒定搅拌。
      2. 混入3毫升甘油
      3. 用ddH 2 O调节体积至10ml
      4. 分装到200μl/管,并储存在-20°C

      5. 加入10μlβME至新鲜等分试样
    4. 缓冲液C(1ml)
      1. 加入10μl1M Tris pH7.6,20μl5M NaCl,100μlTriton X-100和100μl10%SDS。
      2. 加水至1毫升,拌匀,并在4°C储存
    5. 样本缓冲区C(1x)
      用缓冲液C稀释5倍样品缓冲液至1倍
    6. 传输缓冲区

      1. 溶解6.06克Tris和28.8克甘氨酸在1.5升水中
      2. 加1 ml 10%SDS
      3. 将ddH <2> O添加到2L
      4. 在4°C储存,2周内使用
      注意:为了获得最佳和一致的结果,请不要重复使用此传输缓冲区。
    7. HK缓冲区
      1. 将785.2mg KoAc溶于30ml ddH2O中
      2. 加入1毫升的HEPES,pH值为7.2


      3. 调整容量为50毫升,并储存在4°C
    8. TBST
      在1x TBS中0.1%吐温20(v / v)

  3. 库存解决方案
    1. 1M HEPES pH 7.2
      1. 将238.3g HEPES溶于0.5L ddH 2 O中
      2. 用KOH调节pH值至7.2
      3. 将ddH <2> O添加到1L
      4. 在RT
        存储
    2. 10%SDS
      将1g SDS溶解在9ml ddH 2 O中 在RT
      存储
    3. Digitonin股票
      将40毫克的毛地黄皂苷溶解在1毫升DMSO中 在-20°C保存小等份
    4. 胰蛋白酶抑制剂股票
      溶解10毫克胰蛋白酶抑制剂在1毫升B88

      在-20°C保存小份 避免反复冻融
    5. 0.5 M LiCl
      将212毫克LiCl溶解在10毫升B88中 在4°C储存
    6. ATP再生系统(ATP r.s.)
      将2.04克磷酸肌酸,40毫克肌酸激酶,101.44毫克ATP溶于20毫升B88
      将小份储存在-80°C
    7. GTP
      稀释400微升的100毫米GTP与3.6毫升HK缓冲液
      将小份储存在-80℃

致谢

我们感谢加利福尼亚大学伯克利分校细胞培养设施Ann Fisher和Alison Kililea的工作人员。我们也感谢Schekman实验室的过去和现在的成员,特别是梁戈和Yusong Guo在优化这个协议方面进行了有益的讨论。 RS作为霍华德休斯医学研究所和加州大学伯克利分校米勒科学研究所的研究员得到了支持。 LY得到了唐家团契的支持。该协议是从Kim等人2005年描述的以前的工作中修改的。作者声明不存在利益冲突或利益冲突。

参考

  1. Barlowe,C.,Orci,L.,Yeung,T.,Hosobuchi,M.,Hamamoto,S.,Salama,N.,Rexach,MF,Ravazzola,M.,Amherdt,M.and Schekman, )。 COPII:由驱动内质网囊泡出芽的Sec蛋白形成的膜衣。 > Cell 77(6):895-907。
  2. Boyadjiev,SA,Fromme,JC,Ben,J.,Chong,SS,Nauta,C.,Hur,DJ,Zhang,G.,Hamamoto,S.,Schekman,R.,Ravazzola,M.,Orci,L.和Eyaid,W。(2006)。 Cranio-lenticulo-sutural发育异常是由SEC23A突变导致异常内质网 - 高尔基贩运。
  3. Fromme,J.C。和Schekman,R。(2005)。 COPII包裹的囊泡:足够灵活用于大型货物? Curr Opin Cell生物学17(4):345-352。
  4. Gorur,A.,Yuan,L.,Kenny,S. J.,Baba,S.,Xu,K。和Schekman,R.(2017)。 COPII包被膜作为细胞内前胶原蛋白I的转运载体。 J Cell Biol 216(6):1745-1759。
  5. Kaiser,C.A。和Schekman,R。(1990)。 不同组的SEC基因在分泌途径中早期控制运输囊泡的形成和融合。 61(4):723-733。
  6. Kim,J.,Hamamoto,S.,Ravazzola,M.,Orci,L。和Schekman,R。(2005)。 淀粉样蛋白前体蛋白和早老蛋白1的解偶联包装到外壳蛋白复合物II囊泡中< J Biol Chem 280(9):7758-7768。
  7. Novick,P.,Ferro,S.和Schekman,R。(1981)。 酵母分泌途径中的事件顺序 25(2):461-469。
  • English
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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: © 2017 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. Yuan, L., Baba, S., Bajaj, K. and Schekman, R. (2017). Cell-free Generation of COPII-coated Procollagen I Carriers. Bio-protocol 7(22): e2450. DOI: 10.21769/BioProtoc.2450.
  2. Gorur, A., Yuan, L., Kenny, S. J., Baba, S., Xu, K. and Schekman, R. (2017). COPII-coated membranes function as transport carriers of intracellular procollagen I. J Cell Biol 216(6): 1745-1759.
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