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Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana
拟南芥叶绿体内嵌合转运蛋白的定位研究   

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Frontiers in Plant Science
2016

Abstract

In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide and an IEM targeting signal. This protocol makes it possible to investigate the localization of chimeric proteins in chloroplasts using a small number of transgenic plants by using a modified method of chloroplast isolation and fractionation. IEM localization of chimeric proteins can be further assessed by trypsin digestion and alkaline extraction. Here, the localization of the chimeric bicarbonate transporter, designated as SbtAII, is detected by Western blotting using antibodies against Staphylococcal protein A. This protocol is adapted from Uehara et al., 2016.

Keywords: Alkaline (碱性), Arabidopsis (拟南芥), Chimeric bicarbonate transporter (嵌合碳酸氢盐转运蛋白), Chloroplast isolation (叶绿体分离), Chloroplast fractionation (叶绿体分馏), Trypsin (胰蛋白酶)

Background

It has been proposed that the integration of cyanobacterial CO2 concentration mechanisms into chloroplasts is a promising approach to improve photosynthesis in C3 plants. According to theoretical estimations, integration of BicA and SbtA into the chloroplast IEM improves photosynthetic CO2 fixation rates. We examined the integration of nuclear-encoded cyanobacterial bicarbonate transporters, BicA and SbtA, to the IEM of chloroplasts in Arabidopsis. Therefore, we developed a protocol to design chimeric constructs for specific targeting of the IEM and investigate the localization of chimeric proteins in chloroplasts.

Materials and Reagents

  1. Construction of vectors and Arabidopsis transformation
    1. Pipette tips (20 μl, 200 μl, 1,000 μl and 5 ml tips)
    2. 1.5 ml microtubes

  2. Arabidopsis chloroplast isolation
    1. Pipette tips (20 μl, 200 μl, 1,000 μl and 5 ml tips)
    2. 1.5 ml microtubes
    3. Single-edge razor blades
    4. Plastic Petri plates, diameter 150 x 15 mm and diameter 90 x 15 mm
    5. 200 μm nylon mesh cone (Kyoshin Rikoh)
      Note: 100 to 120 mm mesh squares were folded into a cone and stapled to hold its shape (Figure 1).


      Figure 1. Procedure to make the 200 μm mesh cone

    6. Protoplast-rupturing device (Figure 2)
      1. 10 ml disposable syringe (Terumo)
      2. 20 μm nylon mesh (Kyoshin Rikoh)
      3. 10 μm nylon mesh (Kyoshin Rikoh)
      4. Electrical tape, 15-20 mm wide
      Note: Cut off the end of the syringe barrel so that it resembles a hollow tube (Figure 2A). Put the 10 μm mesh on top of the 20 μm mesh and place both over the cut end of the syringe, such that the 10 μm mesh faces the outside and the 20 μm mesh is against the syringe barrel (Figures 2B and 2C). Fix the mesh in place using electrical tape to hold the two layers of mesh to the sides of the syringe, leaving the mesh exposed at the end of the barrel (Figure 2D).


      Figure 2. Procedure to make the protoplast-rupturing device. A. Cut off the end of syringe barrel. B. Syringe barrel, 20 μm mesh (Left), and 10 μm mesh. C. Schematic drawings of making the protoplast-rupturing device. D. Finished product of the protoplast-rupturing device.

    7. Pasteur pipet
    8. Arabidopsis (accession Columbia)
    9. Murashige and Skoog Plant salt mixture (Wako Pure Chemical Industries, catalog number: 392-00591 )
    10. Sucrose (Wako Pure Chemical Industries, catalog number: 196-00015 )
    11. 2-Morpholinoethanesulfonic acid, monohydrate (MES) (DOJINDO, catalog number: 343-01621 )
    12. Agar (Wako Pure Chemical Industries, catalog number: 016-11875 )
    13. Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
    14. Sorbitol (Sigma-Aldrich, catalog number: S1876-1KG )
    15. Calcium chloride dihydrate (CaCl2·2H2O) (Wako Pure Chemical Industries, catalog number: 031-00435 )
    16. Cellulase (Yakult)
    17. Macerozyme (Yakult)
    18. Percoll (GE Healthcare Life Sciences, catalog number: 17089101 )
    19. 1 M 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)-KOH, pH 7.5 and pH 8.0 (HEPES was purchased from DOJINDO, catalog number: 342-01375 )
      Note: The pH of HEPES buffer was adjusted with KOH.
    20. Magnesium chloride hexahydrate (MgCl2·6H2O) (Wako Pure Chemical Industries, catalog number: 135-00165 )
      Note: 1 M magnesium chloride was used in Procedure B.
    21. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Wako Pure Chemical Industries, catalog number: 133-00725 )
      Note: 1 M manganese(II) chloride was used in Procedure B.
    22. Ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
      Note: 0.5 M EDTA was used in Procedure B.
    23. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912-100G )
    24. Tricine (DOJINDO, catalog number: 347-02844 )
      Note: Tricine was directly used in Procedure B.
    25. O,O’-bis(2-aminoethyl)ethyleneglycol-N,N,N’,N’-tetraacetic acid (EGTA) (DOJINDO, catalog number: 346-01312 )
    26. Sodium hydrogen carbonate (NaHCO3) (Wako Pure Chemical Industries, catalog number: 191-01305 )
    27. 0.5x Murashige and Skoog medium (MS medium) (see Recipes)
    28. Digestion buffer (see Recipes)
    29. Digestion enzyme buffer (see Recipes)
    30. 40% (v/v) AT Percoll (see Recipes)
    31. 85% (v/v) AT Percoll (see Recipes)
    32. Gradient buffer (see Recipes)
    33. Protoplast resuspension buffer (see Recipes)
    34. Protoplast breakage buffer (see Recipes)
    35. HEPES-sorbitol buffer, pH 8.0 (see Recipes)

  3. Fractionation of chloroplasts
    1. Pipette tips (20 μl, 200 μl, 1,000 μl and 5 ml tips)
    2. 1.5 ml microtubes
    3. Acetone (Wako Pure Chemical Industries, catalog number: 016-00346 )
      Note: 80% (v/v) acetone was used in Procedure C.
    4. 100% (w/v) trichloroacetic acid (TCA) (Wako Pure Chemical Industries, catalog number: 208-08081 )
    5. Sucrose (Wako Pure Chemical Industries, catalog number: 196-00015 )
    6. Potassium hydroxide (KOH) (Wako Pure Chemical Industries, catalog number: 168-21815 )
    7. Sorbitol (Sigma-Aldrich, catalog number: S1876-1KG )
    8. Ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
      Note: 0.5 M EDTA was used in Procedure C.
    9. Tricine (DOJINDO, catalog number: 347-02844 )
      Note: 1 M Tricine-KOH, pH 7.5, was made for TE/DTT buffer in Procedure C.
    10. Dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 )
      Note: 1 M DTT was used in Procedure C.
    11. Ribonucleic acid, transfer (tRNA) (MP Biomedicals, catalog number: 0215653480 )
    12. 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris) base (Wako Pure Chemical Industries, catalog number: 207-06275 )
      Note: 1 M Tris was used in Procedure C.
    13. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 196-08675 )
      Note: 20% (w/v) SDS was used in Procedure C.
    14. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
      Note: 50% (v/v) glycerol was used in Procedure C.
    15. Saturated bromophenol blue (Wako Pure Chemical Industries, catalog number: 029-02912 )
    16. TE/DTT buffer (see Recipes) containing 1, 0.6, 0.46, 0.2, and 0 M sucrose
    17. SDS-sample buffer (see Recipes)

  4. Trypsin treatment of intact chloroplasts
    1. Pipette tips (20 μl, 200 μl, 1,000 μl and 5 ml tips)
    2. Sorbitol (Sigma-Aldrich, catalog number: S1876-1KG )
    3. Calcium chloride dihydrate (CaCl2·2H2O) (Wako Pure Chemical Industries, catalog number: 031-00435 )
      Note: 1 M calcium chloride was used in Procedure D.
    4. Percoll (GE Healthcare, catalog number: 17089101 )
    5. 1 M 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)-KOH, pH 7.5 and pH 8.0 (HEPES was purchased from DOJINDO, catalog number: 342-01375 )
      Note: The pH of 1 M HEPES buffer was adjusted with KOH.
    6. Magnesium chloride hexahydrate (MgCl2·6H2O) (Wako Pure Chemical Industries, catalog number: 135-00165 )
      Note: 1 M magnesium chloride was used in Procedure D.
    7. Ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
      Note: 0.5 M EDTA was used in Procedure D.
    8. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912-100G )
    9. Dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 )
      Note: 1 M DTT was used in Procedure D.
    10. 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris) base (Wako Pure Chemical Industries, catalog number: 207-06275 )
      Note: 1 M Tris was used in Procedure D.
    11. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 196-08675 )
      Note: 20 % (w/v) SDS was used in Procedure D.
    12. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
      Note: 50% (v/v) glycerol was used in Procedure D.
    13. Saturated bromophenol blue (Wako Pure Chemical Industries, catalog number: 029-02912 )
    14. Trypsin (Sigma-Aldrich, catalog number: T1005 )
      Note: 20 mg/ml trypsin was used in Procedure D.
    15. Nα-Tosyl-L-lysine chloromethyl ketone (TLCK) (Sigma-Aldrich, catalog number: T7254-100MG )
      Note: 50 mg/ml TLCK was used in Procedure D.
    16. Aprotinin (Sigma-Aldrich, catalog number: A3886-1VL )
      Note: 2 mg/ml aprotinin was used in Procedure D.
    17. Phenylmethanesulfonyl fluoride (PMSF) (Wako Pure Chemical Industries, catalog number: 164-12181 )
      Note: 200 mM PMSF was used in Procedure D.
    18. Trypsin inhibitor (Sigma-Aldrich, catalog number: T6522-25MG )
      Note: 10 mg/ml trypsin inhibitor was used in Procedure D.
    19. cOmpleteTM, EDTA-FREE (Roche Diagnostics, catalog number: 11 873 580 001 )
    20. 40% (v/v) AT Percoll (see Recipes)
    21. SDS-sample buffer (see Recipes)
    22. 2x trypsin buffer (see Recipes)
    23. 2x stop buffer (see Recipes)
    24. 1x 40% (v/v) Percoll (see Recipes)
    25. 1x HEPES-sorbitol buffer (see Recipes)
    26. SDS-cOmpleteTM buffer (see Recipes)

  5. Alkaline extraction of chloroplasts
    1. Pipette tips (20 μl, 200 μl, 1,000 μl and 5 ml tips)
    2. Acetone (Wako Pure Chemical Industries, catalog number: 016-00346 )
      Note: 80 % (v/v) acetone was used in Procedure E.
    3. 100% (w/v) trichloroacetic acid (TCA) (Wako Pure Chemical Industries, catalog number: 208-08081 )
    4. Dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 )
      Note: 1 M DTT was used in Procedure E.
    5. Ribonucleic acid, transfer (tRNA) (MP Biomedicals, catalog number: 0215653480 )
    6. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 196-08675 )
      Note: 20 % (w/v) SDS was used in Procedure E.
    7. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
      Note: 50% (v/v) glycerol was used in Procedure E.
    8. Saturated bromophenol blue (Wako Pure Chemical Industries, catalog number: 029-02912 )
    9. Sodium carbonate (Na2CO3), pH 12 (Wako Pure Chemical Industries, catalog number: 199-01585 )
      Note: 0.2 M sodium carbonate was used in Procedure E.
    10. SDS-sample buffer (see Recipes)

  6. Dot blot assay for estimation of protein concentration
    1. Pipette tips (20 μl, 200 μl, 1,000 μl and 5 ml tips)
    2. WhatmanTM 3MM Chromatography paper (GE Healthcare, catalog number: 3030-917 )
    3. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912-100G )
    4. Dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 )
      Note: 1 M DTT was used in Procedure F.
    5. 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris) base (Wako Pure Chemical Industries, catalog number: 207-06275 )
      Note: 1 M Tris was used in Procedure F.
    6. 20% (w/v) sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 196-08675 )
    7. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
      Note: 50% (v/v) glycerol was used in Procedure F.
    8. Saturated bromophenol blue (Wako Pure Chemical Industries, catalog number: 029-02912 )
    9. Coomassie Blue R-250 (Wako Pure Chemical Industries, catalog number: 031-17922 )
    10. Methanol (Wako Pure Chemical Industries, catalog number: 139-01827 )
    11. Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00256 )
    12. Paper towel
    13. SDS-sample buffer (see Recipes)
    14. Coomassie Blue stain (see Recipes)
    15. Coomassie destain solution (see Recipes)

  7. Data analysis
    1. Antibody against the protein A (Sigma-Aldrich, catalog number: P3775 ) (this antibody was used for the detection of chimeric transporter protein tagged with protein A)
    2. Antibody against the large subunit (LSU) of Rubisco (a marker protein of the stroma)
    3. Antibody against Tic (Translocon at the inner envelope membrane of chloroplasts) 110 (a marker protein of the inner envelope membrane)
    4. Antibody against the light-harvesting complex protein (LHCP) (a marker protein of the thylakoid membrane)
    5. Antibody against Toc (Translocon at the outer envelope membrane of chloroplasts) 75 (a marker protein of the outer envelope membrane)
    Note: These four antibodies (40 to 43) are either homemade or provided by other scientists. You may use commercially available antibodies raised against marker proteins for each subcompartment of the chloroplasts.

Equipment

  1. Construction of vectors and Arabidopsis transformation
    1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )

  2. Arabidopsis chloroplast isolation
    1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )
    2. Refrigerator and Freezer
    3. Growth chamber (16 h light/8 h dark, 70-120 μE m-2 sec-1, 22 °C)
    4. 50 ml centrifuge tubes (IWAKI)
    5. Large-capacity centrifuge (TOMY SEIKO, model: Suprema 23 )
    6. Swinging-bucket rotor (TOMY SEIKO, models: TS-33N and B433 )
    7. Swinging-bucket (TOMY SEIKO, model: 3350-G01P )

  3. Fractionation of chloroplasts
    1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )
    2. Refrigerator and Freezer
    3. Homogenizer (IUCHI)
    4. OptimaTM TL Ultracentrifuge (Beckman Coulter)
    5. Angle rotor (Beckman Coulter, model: TLA-100.3 , catalog number: 349490)
    6. 3.5 ml polycarbonate ultracentrifuge tubes (Beckman Coulter, catalog number: 349622 )
    7. Swinging-bucket rotor (Beckman Coulter, model: TLS-55 , catalog number: 346134)
    8. 2.2 ml Ultra-ClearTM centrifuge tubes (Beckman Coulter, catalog number: 347356 )

  4. Trypsin treatment of intact chloroplasts
    1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )

  5. Alkaline extraction of chloroplasts
    1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )
    2. Angle rotor (Beckman Coulter, model: TLA-100.3 , catalog number: 349490)
    3. OptimaTM TL Ultracentrifuge (Beckman Coulter)
    4. 1.5 ml Polyallomer tubes (Beckman Coulter, catalog numbers: 357448 , 355919 )

  6. Dot blot assay for estimation of protein concentrations
    1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )
    2. Labo Shaker (BIO CRAFT, model: BC-740 )

Procedure

  1. Construction of vectors and Arabidopsis transformation
    To deliver a protein of interest to the chloroplast IEM, we have to fuse at least two distinct targeting signals to it. One signal is the transit peptide, and the other is the chloroplast IEM targeting signal (Figure 3). The transit peptide has been known to serve as the targeting signal to the chloroplast stroma and found in the majority of precursor proteins targeted to the chloroplast interior. However, the transit peptide seems to be insufficient to target cargo proteins, in our case the cyanobacterial bicarbonate transporters, to the chloroplast IEM. As an additional IEM targeting signal, we fused the mature portion of the Cor413im1 protein to the chimeric construct shown in Figure 3. This portion can function as the IEM targeting signal in transgenic Arabidopsis (Uehara et al., 2016). Another study has shown that a membrane protein leader can serve as the IEM targeting signal in a transient expression system (Rolland et al., 2016). In addition, it is necessary to add a tag to detect chimeric proteins if specific antibodies are unavailable.
    The length of the transit peptide and the mature portion of Cor413im1 shown in Figure 3 has been already described in detail elsewhere (Okawa et al., 2008 and 2014).


    Figure 3. Construct design for the specific targeting of bicarbonate transporter SbtA II to the chloroplast IEM. Schematic diagram of the chimeric SbtAII construct used in this study. TP indicates transit peptide and serves as the targeting signal to chloroplasts. The Cor413im1 portion of the construct was derived from the mature portion of Cor413im1 and can function as the targeting signal to the chloroplast IEM. The protein A domain of the fusion construct contains two IgG-binding domains from Staphylococcal protein A and serves as the tag for detection of the chimeric protein.

  2. Arabidopsis chloroplast isolation
    We developed a method to isolate sufficient amounts of intact chloroplasts using a smaller number of plants based on the method described in Smith et al. (2002). This method is useful if large numbers of seeds cannot be obtained for the isolation of chloroplasts (e.g., mutants, transgenic lines). Throughout the procedure, samples should be kept on ice unless stated otherwise, and buffers should be chilled prior to experiments.
    1. Sow sterilized seeds on 0.5x MS plates (100 seeds per plate, four plates per isolation, see Recipes) supplemented with 1% sucrose. Allow Arabidopsis plants to grow in growth chambers under a 16 h-light/8 h-dark cycle, 100 μE m-2 sec-1, 22 °C for 14-18 d (Figure 4A). 
    2. Harvest the entire aerial portions of the plants with a razor blade, and place immediately in a 90 mm diameter Petri dish containing 10 ml digestion buffer (see Recipes) on ice. Plants harvested from two plates should be placed into one enzyme digestion reaction so that you need to prepare two 90 mm diameter Petri dishes for four Arabidopsis plates. When all tissue has been harvested, chop the tissue rapidly using a razor blade for up to 1 min (Figure 4B).
      Note: Do not chop for more than 1 min or smash the tissues.
    3. Remove the digestion buffer from the Petri dish. Add 13.5 ml digestion enzyme solution (see Recipes) to the Petri dish, and distribute the tissue uniformly with the fingertips (Figure 4C). Place the Petri dishes into a plastic container, and incubate in a growth chamber for 3 h at 22 °C (light intensity at around 50-100 μE m-2 sec-1).
    4. During digestion, prepare a 40% (20 ml, upper layer):85% (7 ml, lower layer) AT Percoll (see Recipes) step gradient in a 50 ml centrifuge tube and maintain on ice. We use one gradient for two Arabidopsis plates (one enzyme digestion) so that we usually prepare two gradients for one isolation. You may also use smaller Percoll volumes and tubes (Smith et al., 2002).
    5. Harvest the protoplasts through gentle swirling (or gentle agitation) of the plate for 30-60 sec, then filter the medium through a 200 μm mesh cone, placed inside a small funnel, into a 50 ml centrifuge tube on ice. Collect protoplasts from one Petri dish into one 50 ml tube that you need to prepare two tubes. Transfer any tissue from the nylon mesh back into the Petri dish and wash the tissue with 10 ml of ice-cold fresh digestion buffer. Filter the tissue-buffer mixture into the same 50 ml centrifuge tube. Wash the tissue one or two times more to ensure that as many protoplasts are released as possible (Figure 4D).
    6. Centrifuge the protoplasts for 5 min at 100 x g, 4 °C (Figure 4E). Remove and discard the supernatant carefully with an aspirator. Be careful not to disturb the protoplast pellet, as it is very loose.
    7. Resuspend the pellet in 5 ml protoplast resuspension buffer (see Recipes) by gently swirling the pellet while the buffer is dispensed down the side of the tube. After the protoplasts are resuspended, add another 5 ml protoplast resuspension buffer and gently mix (Figure 4F). Centrifuge the protoplasts for 4 min at 100 x g, 4 °C (Figure 4G).
    8. Remove the supernatant and resuspend the pellet in 5 ml protoplast breakage buffer (see Recipes). After the protoplasts are resuspended, add another 5 ml protoplast breakage buffer and gently mix (Figure 4H). Immediately transfer the resuspended protoplast pellet into the barrel of the protoplast-rupturing device. Holding the end of the device over a 50 ml centrifuge tube on ice, carefully replace the plunger, and gently and firmly force the suspension through the layers of mesh (Figure 4I). Repeat this procedure once.
    9. Quickly and carefully layer the 10 ml broken protoplasts onto the AT Percoll step gradient (Figure 4J). Centrifuge in a swinging-bucket rotor for 10 min at 2,500 x g, 4 °C, with the brake off (Figure 4K). Following centrifugation, there should be two visible green bands in the gradient: an upper band of broken chloroplasts at the protoplast breakage buffer/40% Percoll interface, and a lower band of intact chloroplasts at 40%/85% Percoll interface. Remove the load zone and the upper band of broken chloroplasts by aspiration. Harvest the lower band using a Pasteur pipet, and dilute the intact chloroplasts with 40-45 ml HEPES-sorbitol buffer (see Recipes), pH 8.0, in a 50 ml centrifuge tube (Figure 4L). Intact chloroplasts from the two AT Percoll gradients should be combined into one tube.
    10. Centrifuge the diluted intact chloroplasts for 5 min at 700 x g, 4 °C (Figure 4M). Carefully decant the supernatant, and discard all excess buffer without disrupting the pellet. Resuspend the pellet in a small volume (200-300 μl) of HEPES-sorbitol buffer, pH 8.0 (Figure 4N).
    11. Dilute 5 μl of the chloroplast resuspension into 995 μl 80% acetone. Mix vigorously and microcentrifuge at ~15,000 x g for 2 min to remove the protein precipitate. Measure the A652 of the supernatant against an 80% acetone blank. Calculate the chlorophyll concentration (mg/ml) by using the following equation (Smith et al., 2002):
      (A652/36) x 200
      to compensate for the dilution factor. Adjust the chlorophyll concentration to 1 mg chlorophyll/ml using HEPES-sorbitol buffer. The prepared chloroplasts should be used immediately for Procedures C and D, while chloroplasts for procedure E can be stored at -80 °C.


      Figure 4. Overview of Arabidopsis chloroplasts isolation (Procedure B)

  3. Fractionation of chloroplasts
    1. Centrifuge 0.5 ml of 1 mg chlorophyll/ml chloroplasts prepared in Step B11 for 5 min at 700 x g, 4 °C. Remove the supernatant and measure the volume of supernatant by pipetting. Resuspend the chloroplast pellet in TE/DTT buffer (see Recipes) containing 0.6 M sucrose to a concentration of 1 mg/ml chlorophyll (Figure 5A). Let stand for 10 min on ice. Freeze the chloroplast suspension for 1-2 h at -20 °C. If you do not proceed to the fractionation stage immediately, it is possible to keep the frozen chloroplast suspension at -80 °C until use.
    2. Thaw the suspension and dilute with 3 volumes of TE/DTT buffer. To collect the total chloroplast proteins, take 5 μl of chloroplast suspension into 1.5 ml microtubes before the addition of TE/DTT. Homogenize with 20 strokes in a Dounce (or Potter) homogenizer with a tight pestle (Figure 5B). Transfer the suspension into 3.5 ml polycarbonate ultracentrifuge tubes. Ultracentrifuge the lysed chloroplasts in a fixed angle rotor for 1 h at 40,000 x g, 4 °C (Figure 5C).
    3. Remove the brownish supernatant containing the stromal content using a pipette and store at -80 °C (Figure 5D). Resuspend the membrane pellet in TE/DTT buffer containing 0.2 M sucrose to a concentration of 1 mg chlorophyll/ml by pipetting (Figure 5D) and homogenization (Figure 5E, 20 strokes in a Dounce homogenizer).
    4. Set up a sucrose step gradient consisting of 1 ml of 1 M sucrose, and 0.7 ml of 0.46 M sucrose in 2.2 ml polyallomer tubes. Layer the membrane suspension (0.2 M sucrose) onto the top (Figure 5F and Figure 6A). Apply 0.5 ml of membrane suspension on each gradient. Ultracentrifuge the samples for 1.5 h at 270,000 x g, 4 °C, in a swinging-bucket rotor using low acceleration and deceleration rates (Figure 5G). Collect the interface from each step of the gradient (Figure 5H and Figure 6B). The upper interface (0.2:0.46 M sucrose) contains residual stroma and plastoglobules. The middle interface (0.46:1 M sucrose) is highly enriched in envelope membranes. The thylakoid membranes form a tight pellet at the bottom of the tube.
    5. Precipitate the stromal proteins (collected in Step C2) by adding 100% trichloroacetic acid (TCA) to give a final concentration of 10% and tRNA to give a final concentration of 10 μg/ml. Incubate for 60 min or longer on ice. Collect the TCA precipitate by centrifugation for 30 min at ~20,000 x g, 4 °C. Wash the pellet with 1 ml of 80% acetone and centrifuge for 15 min at ~20,000 x g, 4 °C. Resuspend the pellet directly in 200 μl SDS-sample buffer (see Recipes).
    6. Precipitate the total chloroplast proteins (5 μl collected in Step C2) by adding 1 ml of 80% acetone and vortex. Keep the tube on ice for 30 min and centrifuge for 30 min at ~20,000 x g, 4 °C. Resuspend the pellet directly in 50 μl SDS-sample buffer.
    7. To collect the envelope membranes, dilute the 0.46:1 M sucrose interface fraction with 3 to 5 volumes of TE/DTT buffer and centrifuge in a fixed angle rotor for 1 h at 270,000 x g, 4 °C (Figure 5J). Remove the supernatant with a pipette and discard. Resuspend the pellets in 30 μl of SDS-sample buffer for analysis (Figure 5K).
    8. Thylakoid pellets are suspended in 0.3 ml SDS-sample buffer (Figure 5H).
    9. After the quantification of proteins in each fraction, analyze total chloroplast (3 μg), stroma (3 μg), envelope (1 μg), and thylakoid (1.5 μg) fractions by SDS-PAGE.


      Figure 5. Overview of chloroplast fractionation (Procedure C)


      Figure 6. Schematic drawings of sucrose density gradient ultracentrifugation for the separation of envelope and thylakoid membrane (Step C4). This experiment uses 2.2 ml Ultra-ClearTM centrifuge tubes. A and B represent sucrose density gradients before and after ultracentrifugation, respectively.

  4. Trypsin treatment of intact chloroplasts
    Trypsin is capable of permeating the outer envelope membrane (OEM) but not the inner envelope membrane (IEM) of intact chloroplasts (Jackson et al., 1998; Inaba et al., 2003). Therefore, proteins localized to the OEM, such as Toc75, are sensitive to trypsin treatment. This method allows us to determine whether a protein of interest localizes to the OEM or IEM of chloroplasts.
    1. Dilute 25 μl of 1 mg chlorophyll/ml intact chloroplasts (Step B11) with 125 μl of HEPES-sorbitol buffer.
    2. Add 150 μl of 2x trypsin buffer (see Recipes), mix gently, and incubate for 30-40 min on ice.
    3. Stop the reaction by adding 300 μl of 2x stop buffer (see Recipes). Mix very carefully by inverting the tube several times and incubate on ice for 10 min. After incubation on ice, isolate the chloroplasts by centrifugation through a 1x 40% Percoll (see Recipes) for 5 min at 2,500 x g, 4 °C.
    4. Carefully aspirate the supernatant and 1x 40% Percoll, containing some broken chloroplasts.
    5. Resuspend the chloroplast pellet in 500 μl of 1x HEPES-sorbitol buffer (see Recipes). Centrifuge the chloroplasts for 2 min at 2,500 x g, 4 °C. Remove the supernatant and resuspend the pellet in 50 μl of SDS-cOmpleteTM buffer (see Recipes).

  5. Alkaline extraction of chloroplasts
    To investigate whether a protein of interest is integrated into the envelope membranes, chloroplasts can be subjected to alkaline extraction. If the protein is integrated into the envelope membranes, it should be recovered in the pellet fraction after alkaline extraction and subsequent ultracentrifugation. Peripherally associated IEM proteins, such as Tic22, are released from the IEM during the extraction (Kouranov et al., 1998).
    1. Dilute 25 μl of 1 mg chlorophyll/ml chloroplasts (Step B11) into 1 ml of 0.2 M Na2CO3, pH 12, in a 1.5 ml polyallomer tube and incubate for 10 min on ice.
    2. Ultracentrifuge the mixture in a fixed angle rotor for 15 min at 100,000 x g, 4 °C. Carefully collect the supernatant containing the stroma and transfer to a fresh tube. Resuspend the membrane pellet in 80 μl SDS-sample buffer. Dissolve the pellet completely.
    3. Precipitate the alkaline supernatant by adding 100% (w/v) TCA to a final concentration of 10% (w/v) and tRNA to a final concentration of 10 μg/ml. Incubate for 30 to 60 min on ice.
    4. Collect the TCA precipitate by centrifuging for 30 min at ~20,000 x g, 4 °C. Wash the pellet with 1 ml of 80% acetone and centrifuge for 15 min at ~20,000 x g, 4 °C.
    5. Resuspend the pellet directly in 80 μl SDS-sample buffer. Dissolve the pellet completely.

  6. Dot blot assay for estimation of protein concentrations
    Estimation of concentrations of samples from Procedures C and D are performed as follows:
    1. Cut a square of WhatmanTM 3MM Chromatography paper (3MM paper) and place it on a clean sheet of paper towel.
    2. Mark the concentration of BSA standards and each sample to be tested on 3MM paper with a pencil (see Figure 7).
    3. Dilute the 2.0 mg/ml bovine serum albumin (BSA) with SDS-sample buffer to make BSA standards (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 mg/ml). Dilute 3 μl of each sample to be tested with 3 μl SDS-sample buffer. If the protein concentration is expected to be high, make a serial dilution as shown in Figure 7.
    4. Gently dot 2 μl of the BSA standards and each sample onto the 3MM paper with a pipette.
    5. Dry the 3MM paper at room temperature for at least 20 min.
    6. Stain on a shaker with Coomassie Blue stain for 30 min.
    7. Collect Coomassie Blue stain as much as possible (reusable).
    8. Destain the paper on a shaker with Coomassie destain solution. Change the destain solution every 20 min. Destain until the background is clear.
    9. Dry the 3MM paper on a paper towel.
    10. Estimate the concentration of each sample by comparing the intensity of the blot color to the BSA standards.

Data analysis

Determination of protein concentration and SDS-PAGE

  1. Compare the strength of each signal to the BSA standards and determine the concentration of protein (Figure 7: e.g., the signal strength of the 1/2-diluted Cp spot is almost equivalent to that of the 0.6 mg/ml BSA standard spot).
  2. Prepare samples based on quantification of proteins in each fraction. Sample volumes can be adjusted using SDS-sample buffer.
    Note: In most cases, a portion of each fraction is sufficient to detect the protein of interest. The protein ratio of Cp:Str:Env:Thy (prepared in Procedure C) should be consistently 3:3:1:1.5.


    Figure 7. Dot blot quantification of protein concentration. The upper portion indicates the staining of BSA standards (STD). Isolated chloroplasts (Cp) were fractionated into stroma (Str), envelope membrane (Env), and thylakoid membrane (Thy) fractions in Procedure C. Each sample was diluted 2, 4, 8, 16, 32 times with SDS-sample buffer.

  3. The proteins recovered in each fraction (Procedures C, D, and E) were resolved by SDS-PAGE and analyzed by Western blotting.
  4. Investigate the localization of chimeric proteins within the chloroplasts. As an example, we used SbtAII. The purity of each fraction was confirmed using marker proteins such as LSU (stroma), Tic110 (envelope membrane), and LHCP (thylakoid membrane). The SbtAII fusion protein was localized in chloroplasts (Figure 8, lane Cp). Furthermore, the SbtAII fusion protein was found to be highly enriched in the envelope fraction (Figure 8, lane Env).


    Figure 8. Localization of chimeric protein SbtAII in Arabidopsis chloroplasts (Procedure C). Isolated chloroplasts (Cp) were fractionated into stroma (Str), envelope membrane (Env), and thylakoid membrane (Thy) fractions. The protein ratio of Cp:Str:Env:Thy used in these analyses was consistently 3:3:1:1.5. Each fraction was western blotted with antibodies against protein A, LSU, Tic110 and LHCP.

  5. Investigate whether SbtAII is an outer or inner envelope membrane protein. Trypsin permeates the outer envelope membrane, but not the inner envelope membrane, of intact chloroplasts. As expected, the OEM protein Toc75 was digested by trypsin (Figure 9, Toc75). In contrast, chimeric SbtAII (Figure 9, Protein A) and the IEM protein, Tic110 (Figure 9, Tic110), are resistant to trypsin, indicating that the chimeric protein is localized to the IEM of chloroplasts.


    Figure 9. Trypsin sensitivity of chimeric SbtA protein in chloroplasts (Procedure D). The protease sensitivity of the outer envelope membrane protein, Toc75, was included as a positive control. The protease resistance of the inner envelope membrane protein, Tic110, was also included as a control.

  6. Investigate whether SbtAII is integrated into the IEM, or is peripherally associated with the IEM. Tic110 is an integral IEM protein and is shown as a positive control. SbtAII was resistant to alkaline extraction (Figure 10), indicating that SbtAII is an integral membrane protein in the chloroplast IEM.


    Figure 10. Localization of chimeric SbtA II protein in the soluble and membrane fractions of chloroplasts (Procedure E). Chloroplasts treated with alkaline solution were separated into insoluble (P) and soluble (S) fractions. The insoluble protein, Tic110, and the soluble protein, LSU, are included as controls.

Recipes

  1. 0.5x Murashige and Skoog medium (MS medium) (60 ml per plate)
    0.23 g Murashige and Skoog Plant salt mixture (final 0.5x)
    1 g sucrose (final 1% (w/v))
    0.05 g MES (final 2.3 mM)
    ddH2O to 100 ml, adjust pH to 5.7-5.8 using 1 N KOH
    After adjusting, add 0.5 g agar (final 0.5%) and autoclave
  2. Digestion buffer (store up to 2 weeks at 4 °C)
    1.56 g MES (final 20 mM)
    29.13 g sorbitol (final 400 mM)
    200 μl 1 M CaCl2 (final 0.5 mM)
    ddH2O to 400 ml, adjust pH to 5.2 using 1 N KOH
  3. Digestion enzyme buffer
    Dissolve 0.6 g cellulase and 0.12 g macerozyme in 30 ml digestion buffer
    Centrifuge for 10 min at 2,000 x g to pellet insoluble materials
    Use supernatant immediately
  4. 40% (v/v) AT Percoll (store up to 1 month at -20 °C, thaw immediately before use)
    40 ml Percoll (final 40% (v/v))
    50 ml gradient buffer (final 50% (v/v))
    10 ml ddH2O
  5. 85% (v/v) AT Percoll (store up to 1 month at -20 °C, thaw immediately before use)
    42.5 ml Percoll (final 85% (v/v))
    2.5 ml 1 M HEPES-KOH, pH 7.5 (final 50 mM)
    3.01 g sorbitol (final 330 mM)
    Add ddH2O to 50 ml
  6. Gradient buffer (store up to 2 weeks at 4 °C)
    10 ml 1 M HEPES-KOH, pH 7.5 (final 100 mM)
    12.04 g sorbitol (final 660 mM)
    200 μl 1 M MgCl2 (final 2 mM)
    200 μl 1 M MnCl2 (final 2 mM)
    800 μl 0.5 M EDTA (final 4 mM)
    0.2 g BSA (final 0.2% (w/v)) (add immediately before use)
    ddH2O to 100 ml
  7. Protoplast resuspension buffer (store up to 2 weeks at 4 °C)
    0.39 g MES (final 20 mM)
    7.29 g sorbitol (final 400 mM)
    50 μl 1 M CaCl2 (final 0.5 mM)
    ddH2O to 100 ml, adjust pH to 6.0 using 1 N KOH
  8. Protoplast breakage buffer (store up to 2 weeks at 4 °C)
    0.36 g Tricine (final 20 mM)
    5.47 g sorbitol (final 300 mM)
    1 ml 0.5 M EDTA (final 5 mM)
    0.19 g EGTA (final 5 mM)
    0.084 g NaHCO3 (final 10 mM)
    0.1 g BSA (final 0.1% (w/v)) (add immediately before use)
    ddH2O to 100 ml, adjust pH to 8.4 using 1 N KOH
  9. HEPES-sorbitol buffer, pH 8.0 (store up to 2 weeks at 4 °C)
    15 ml 1 M HEPES-KOH, pH 8.0 (final 50 mM)
    18.06 g sorbitol (final 330 mM)
    ddH2O to 300 ml
  10. TE/DTT buffer containing 1, 0.6, 0.46, 0.2 and 0 M sucrose (store up to 2 weeks at 4 °C)
    0.25 ml 1 M Tricine-KOH, pH 7.5 (final 50 mM)
    20 μl 0.5 M EDTA (final 2 mM)
    5 μl 1 M DTT (final 1 mM) (add immediately before use)
    1.712, 1.046, 0.787, 0.343 g sucrose (final 1, 0.6, 0.46, 0.2, and 0 M)
    ddH2O to 5 ml
  11. SDS-sample buffer (store at -20 °C until used)
    0.35 ml 1 M Tris base (final 350 mM)
    0.25 ml 20% (w/v) SDS (final 5% (w/v))
    80 μl 1 M DTT (final 80 mM)
    0.15 ml 50% (v/v) glycerol (final 7.5% (v/v))
    40 μl saturated bromophenol blue (final 1% (v/v))
    0.13 ml ddH2O
  12. 2x trypsin buffer
    8 μl 1 M MgCl2 (final 8 mM)
    0.2 μl 1 M CaCl2 (final 0.2 mM)
    HEPES-sorbitol buffer to 1 ml
    Add 6.6 μl 20 mg/ml trypsin in 300 μl 2x trypsin buffer
  13. 2x stop buffer
    Add 2 μl 50 mg/ml TLCK
    2 μl 2 mg/ml aprotinin
    10 μl 200 mM PMSF
    20 μl 10 mg/ml trypsin inhibitor
    20 μl 0.5 M EDTA
    in 1 ml HEPES-sorbitol buffer
  14. 1x 40% Percoll
    Add 1 μl 50 mg/ml TLCK
    1 μl 2 mg/ml aprotinin
    5 μl 200 mM PMSF
    10 μl 10 mg/ml trypsin inhibitor
    10 μl 0.5 M EDTA
    in 1 ml 40% Percoll
  15. 1x HEPES-sorbitol buffer
    Add 1 μl 50 mg/ml TLCK
    1 μl 2 mg/ml aprotinin
    5 μl 200 mM PMSF
    10 μl 10 mg/ml trypsin inhibitor
    10 μl 0.5 M EDTA
    in 1 ml HEPES-sorbitol buffer
  16. SDS-cOmpleteTM buffer
    10 μl 0.5 M EDTA (final 10 mM)
    20 μl 25x cOmpleteTM EDTA-free (final 1x)
    470 μl SDS-sample buffer
  17. Coomassie Blue stain (store at room temperature)
    0.125 g Coomassie Blue R250 (final 0.125% (w/v))
    50 ml methanol (final 50% (v/v))
    10 ml acetic acid (final 10% (v/v))
    ddH2O to 100 ml
  18. Coomassie destain solution (store at room temperature)
    45 ml methanol (final 45% (v/v))
    10 ml acetic acid (final 10% (v/v))
    45 ml ddH2O

Acknowledgments

This protocol was modified from previously published works (Uehara et al., 2016). This work was supported by JSPS KAKENHI Grant Number 17J06506 (to S.U.), 17K07762 (to Y.I.I.) and 15K07843 (to T.I.). The authors declare no conflicts of interest or competing interests.

References

  1. Inaba, T., Li, M., Alvarez-Huerta, M., Kessler, F. and Schnell, D. J. (2003). atTic110 functions as a scaffold for coordinating the stromal events of protein import into chloroplasts. J Biol Chem 278(40): 38617-38627.
  2. Jackson, D. T., Froehlich, J. E. and Keegstra, K. (1998). The hydrophilic domain of Tic110, an inner envelope membrane component of the chloroplastic protein translocation apparatus, faces the stromal compartment. J Biol Chem 273(26): 16583-16588.
  3. Kouranov, A., Chen, X., Fuks, B. and Schnell, D. J. (1998). Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane. J Cell Biol 143(4): 991-1002.
  4. Okawa, K., Inoue, H., Adachi, F., Nakayama, K., Ito-Inaba, Y., Schnell, D. J., Uehara, S., and Inaba, T. (2014). Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments. J Exp Bot 65(18): 5257-5265.
  5. Okawa, K., Nakayama, K., Kakizaki, T., Yamashita, T., and Inaba, T. (2008). Identification and characterization of cor413im proteins as novel components of the chloroplast inner envelope. Plant Cell Environ 31(10): 1470-1483.
  6. Rolland, V., Badger, M. R. and Price, G. D. (2016). Redirecting the cyanobacterial bicarbonate transporters BicA and SbtA to the chloroplast envelope: Soluble and membrane cargos need different chloroplast targeting signals in plants. Front Plant Sci 7: 185.
  7. Smith, M. D., Schnell, D. J., Fitzpatrick, L. and Keegstra, K. (2003). In vitro analysis of chloroplast protein import. Curr Protoc Cell Biol Chapter 11: Unit11 16.
  8. Uehara, S., Adachi, F., Ito-Inaba, Y. and Inaba, T. (2016). Specific and efficient targeting of cyanobacterial bicarbonate transporters to the inner envelope membrane of chloroplasts in Arabidopsis. Front Plant Sci 7: 16.

简介

在这个协议中,我们描述了一种设计嵌合蛋白的方法,用于特异性靶向拟南芥叶绿体的内包膜(IEM)并通过生化分析确定它们的定位。 叶绿体IEM的特异性靶向可通过将感兴趣的蛋白质与转运肽和IEM靶向信号融合来实现。 这个协议使得有可能使用少量的转基因植物,通过使用修改的叶绿体分离和分离方法来研究嵌合蛋白在叶绿体中的定位。 嵌合蛋白的IEM定位可以通过胰蛋白酶消化和碱性提取进一步评估。 在此,称为SbtAII的嵌合碳酸氢根转运蛋白的定位通过使用针对葡萄球菌蛋白A的抗体进行蛋白质印迹来检测。该方案改编自上原等人,2016年


【背景】有人提出将蓝藻CO 2浓度机制整合到叶绿体中是改善C 3+植物光合作用的有希望的方法。 根据理论估计,将BicA和SbtA整合到叶绿体IEM中可以提高光合CO 2固定率。 我们研究了核编码的蓝细菌碳酸氢盐转运蛋白BicA和SbtA与拟南芥叶绿体的IEM的整合。 因此,我们制定了一个协议,设计嵌合构造为特定目标的IEM和调查嵌合蛋白在叶绿体中的定位。

关键字:碱性, 拟南芥, 嵌合碳酸氢盐转运蛋白, 叶绿体分离, 叶绿体分馏, 胰蛋白酶

材料和试剂

  1. 载体构建和拟南芥转化
    1. 移液器吸头(20μl,200μl,1,000μl和5ml吸头)
    2. 1.5毫升微管

  2. 拟南芥叶绿体分离
    1. 移液器吸头(20μl,200μl,1,000μl和5ml吸头)
    2. 1.5毫升microtubes
    3. 单刃剃须刀片
    4. 塑料培养皿,直径150 x 15毫米,直径90 x 15毫米
    5. 200μm尼龙网眼锥(Kyoshin Rikoh)
      注:100至120毫米的网格正方形被折叠成一个圆锥体并钉住以保持其形状(图1)。


      图1.制作200μm网状锥体的步骤

    6. 原生质体破裂装置(图2)
      1. 10毫升一次性注射器(Terumo)
      2. 20μm尼龙网(Kyoshin Rikoh)
      3. 10μm尼龙网(Kyoshin Rikoh)
      4. 电工胶带,宽15-20毫米
      注意:切断注射器筒的末端,使其看起来像中空管(图2A)。将10微米网格放在20微米网格的顶部,并将其放在注射器的切割端上,使得10微米网格面向外部,并且20微米网格靠着注射器筒(图2B和2C)。使用电工胶带将网状物固定到位,使两层网状物保持在注射器的两侧,使网状物暴露在筒的末端(图2D)。


      图2.制备原生质体破裂装置的步骤A.切断注射器筒的末端。 B. 20μm网眼(左)和10μm网眼的注射器针筒。 C.制造原生质体破裂装置的示意图。 D.原生质体破裂装置的成品。

    7. 巴斯德吸管
    8. 拟南芥(加入哥伦比亚)
    9. Murashige和Skoog植物盐混合物(Wako Pure Chemical Industries,目录号:392-00591)
    10. 蔗糖(Wako Pure Chemical Industries,目录号:196-00015)
    11. 2-吗啉代乙烷磺酸一水合物(MES)(DOJINDO,目录号:343-01621)
    12. 琼脂(Wako Pure Chemical Industries,产品目录号:016-11875)
    13. 氢氧化钾(KOH)(Wako Pure Chemical Industries,目录号:168-21815)
    14. 山梨醇(Sigma-Aldrich,目录号:S1876-1KG)
    15. 氯化钙二水合物(CaCl 2•2H 2 O)(Wako Pure Chemical Industries,目录号:031-00435)
    16. 纤维素酶(Yakult)
    17. Macerozyme(Yakult)
    18. Percoll(GE Healthcare Life Sciences,目录号:17089101)
    19. 1 H 2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(HEPES)-KOH,pH7.5和pH8.0(HEPES购自DOJINDO,目录号:342-01375)
      注意:用KOH调节HEPES缓冲液的pH值。
    20. 氯化镁六水合物(MgCl 2•6H 2 O)(Wako Pure Chemical Industries,目录号:135-00165)
      注:步骤B中使用1M氯化镁。
    21. 氯化锰(II)四水合物(MnCl 2•4H 2 O)(Wako Pure Chemical Industries,目录号:133-00725)
      注意:步骤B中使用1M氯化锰(II)。
    22. 乙二胺四乙酸(EDTA)(DOJINDO,目录号:345-01865)
      注意:步骤B中使用0.5M EDTA。
    23. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912-100G)
    24. Tricine(DOJINDO,目录号:347-02844)
      注意:Tricine直接用于程序B.
    25. (2-氨乙基)乙二醇-N,N,N',N' - 四乙酸(EGTA)(DOJINDO,目录号:346- 01312)
    26. 碳酸氢钠(NaHCO 3)(Wako Pure Chemical Industries,目录号:191-01305)
    27. 0.5倍Murashige和Skoog培养基(MS培养基)(见食谱)
    28. 消化缓冲液(见食谱)
    29. 消化酶缓冲液(见食谱)
    30. 40%(v / v)AT Percoll(见食谱)
    31. 85%(v / v)AT Percoll(见食谱)
    32. 渐变缓冲区(见食谱)
    33. 原生质体重悬缓冲液(见食谱)
    34. 原生质体破损缓冲液(见食谱)
    35. HEPES-山梨糖醇缓冲液,pH8.0(见食谱)

  3. 叶绿体分馏
    1. 移液器吸头(20μl,200μl,1,000μl和5ml吸头)
    2. 1.5毫升microtubes
    3. 丙酮(Wako Pure Chemical Industries,目录号:016-00346)
      注意:步骤C中使用80%(v / v)丙酮。
    4. 100%(w / v)三氯乙酸(TCA)(Wako Pure Chemical Industries,目录号:208-08081)
    5. 蔗糖(Wako Pure Chemical Industries,目录号:196-00015)
    6. 氢氧化钾(KOH)(Wako Pure Chemical Industries,目录号:168-21815)
    7. 山梨醇(Sigma-Aldrich,目录号:S1876-1KG)
    8. 乙二胺四乙酸(EDTA)(DOJINDO,目录号:345-01865)
      注:步骤C中使用0.5M EDTA。
    9. Tricine(DOJINDO,目录号:347-02844)
      注意:在程序C中,为TE / DTT缓冲液制备1M Tricine-KOH,pH7.5。
    10. 二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:041-08976)
      注意:程序C中使用了1 M DTT
    11. 核糖核酸转移(tRNA)(MP Biomedicals,产品目录号:0215653480)
    12. 2-氨基-2-羟甲基-1,3-丙二醇(Tris)碱(Wako Pure Chemical Industries,目录号:207-06275)
      注意:步骤C中使用1M Tris。
    13. 十二烷基硫酸钠(SDS)(Wako Pure Chemical Industries,目录号:196-08675)
      注意:程序C中使用20%(w / v)SDS。
    14. 甘油(Wako Pure Chemical Industries,目录号:075-00616)
      注:步骤C中使用50%(v / v)甘油。
    15. 饱和溴酚蓝(Wako Pure Chemical Industries,目录号:029-02912)
    16. 含有1,0.6,0.46,0.2和0M蔗糖的TE / DTT缓冲液(参见配方)
    17. SDS样品缓冲液(见食谱)

  4. 胰蛋白酶处理完整的叶绿体
    1. 移液器吸头(20μl,200μl,1,000μl和5ml吸头)
    2. 山梨醇(Sigma-Aldrich,目录号:S1876-1KG)
    3. 氯化钙二水合物(CaCl 2•2H 2 O)(Wako Pure Chemical Industries,目录号:031-00435)
      注意:步骤D中使用1M氯化钙。
    4. Percoll(GE Healthcare,目录号:17089101)
    5. 1 H 2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(HEPES)-KOH,pH7.5和pH8.0(HEPES购自DOJINDO,目录号:342-01375)
      注意:用KOH调节1M HEPES缓冲液的pH。
    6. 氯化镁六水合物(MgCl 2•6H 2 O)(Wako Pure Chemical Industries,目录号:135-00165)
      注意:步骤D中使用1M氯化镁。
    7. 乙二胺四乙酸(EDTA)(DOJINDO,目录号:345-01865)
      注意:步骤D中使用0.5M EDTA。
    8. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912-100G)
    9. 二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:041-08976)
      注意:程序D中使用了1 M DTT
    10. 2-氨基-2-羟甲基-1,3-丙二醇(Tris)碱(Wako Pure Chemical Industries,目录号:207-06275)
      注意:步骤D中使用1M Tris。
    11. 十二烷基硫酸钠(SDS)(Wako Pure Chemical Industries,目录号:196-08675)
      注意:程序D中使用了20%(w / v)的SDS。
    12. 甘油(Wako Pure Chemical Industries,目录号:075-00616)
      注:步骤D中使用50%(v / v)甘油。
    13. 饱和溴酚蓝(Wako Pure Chemical Industries,目录号:029-02912)
    14. 胰蛋白酶(Sigma-Aldrich,目录号:T1005)
      注意:步骤D中使用20mg / ml胰蛋白酶。
    15. α-甲苯磺酰-L-赖氨酸氯甲基酮(TLCK)(Sigma-Aldrich,目录号:T7254-100MG)
      注:步骤D中使用50mg / ml TLCK。
    16. 抑肽酶(Sigma-Aldrich,目录号:A3886-1VL)
      注:步骤D中使用2mg / ml抑肽酶。
    17. 苯基甲磺酰氟(PMSF)(Wako Pure Chemical Industries,目录号:164-12181)
      注意:步骤D中使用200mM PMSF。
    18. 胰蛋白酶抑制剂(Sigma-Aldrich,目录号:T6522-25MG)
      注:步骤D中使用10mg / ml胰蛋白酶抑制剂。
    19. cOmplete TM TM,EDTA-FREE(Roche Diagnostics,目录号:11 873 580 001)
    20. 40%(v / v)AT Percoll(见食谱)
    21. SDS样品缓冲液(见食谱)
    22. 2x胰蛋白酶缓冲液(见食谱)
    23. 2个停止缓冲区(见食谱)
    24. 1倍40%(v / v)Percoll(见食谱)
    25. 1倍HEPES山梨糖醇缓冲液(见食谱)
    26. SDS-cOmplete 缓冲液(见食谱)

  5. 碱性提取叶绿体
    1. 移液器吸头(20μl,200μl,1,000μl和5ml吸头)
    2. 丙酮(Wako Pure Chemical Industries,目录号:016-00346)
      注:步骤E中使用80%(v / v)丙酮。
    3. 100%(w / v)三氯乙酸(TCA)(Wako Pure Chemical Industries,目录号:208-08081)
    4. 二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:041-08976)
      注意:程序E中使用了1 M DTT
    5. 核糖核酸转移(tRNA)(MP Biomedicals,产品目录号:0215653480)
    6. 十二烷基硫酸钠(SDS)(Wako Pure Chemical Industries,目录号:196-08675)
      注意:步骤E中使用20%(w / v)SDS。
    7. 甘油(Wako Pure Chemical Industries,目录号:075-00616)
      注:步骤E中使用50%(v / v)甘油。
    8. 饱和溴酚蓝(Wako Pure Chemical Industries,目录号:029-02912)
    9. 碳酸钠(Na 2 CO 3),pH 12(Wako Pure Chemical Industries,目录号:199-01585)
      注意:步骤E中使用0.2M碳酸钠。
    10. SDS-样品缓冲液(见食谱)

  6. 用于评估蛋白质浓度的斑点印迹分析
    1. 移液器吸头(20μl,200μl,1,000μl和5ml吸头)
    2. Whatman TM 3MM色谱纸(GE Healthcare,目录号:3030-917)
    3. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912-100G)
    4. 二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:041-08976)
      注意:程序F中使用了1 M DTT
    5. 2-氨基-2-羟甲基-1,3-丙二醇(Tris)碱(Wako Pure Chemical Industries,目录号:207-06275)
      注意:步骤F中使用1M Tris。
    6. 20%(w / v)十二烷基硫酸钠(SDS)(Wako Pure Chemical Industries,目录号:196-08675)
    7. 甘油(Wako Pure Chemical Industries,目录号:075-00616)
      注:步骤F中使用50%(v / v)甘油。
    8. 饱和溴酚蓝(Wako Pure Chemical Industries,目录号:029-02912)
    9. 考马斯蓝R-250(Wako Pure Chemical Industries,目录号:031-17922)
    10. 甲醇(Wako Pure Chemical Industries,目录号:139-01827)
    11. 乙酸(Wako Pure Chemical Industries,目录号:017-00256)
    12. 纸巾
    13. SDS样品缓冲液(见食谱)
    14. 考马斯蓝染色(见食谱)
    15. 考马斯亮染液(见食谱)

  7. 数据分析
    1. 抗蛋白质A的抗体(Sigma-Aldrich,目录号:P3775)(该抗体用于检测用蛋白A标记的嵌合转运蛋白)
    2. 抗Rubisco的大亚基(LSU)(基质的标记蛋白)的抗体
    3. 抗Tic的抗体(叶绿体内膜包膜上的转座子)110(内包膜的标记蛋白)
    4. 抗捕光复合蛋白(LHCP)(类囊体膜的标志蛋白)的抗体
    5. 抗Toc抗体(叶绿体外膜包裹的转座子)75(外包膜的标记蛋白)
    注意:这四种抗体(40到43)是自制的或由其他科学家提供的。您可以使用针对叶绿体每个小分区的标记蛋白产生的商业抗体。

设备

  1. 载体构建和拟南芥转化
    1. 移液器(Gilson,型号:P20,P200,P1000和P5000,目录号:F123600,F123601,F123602和F123603)

  2. 拟南芥叶绿体分离
    1. 移液器(Gilson,型号:P20,P200,P1000和P5000,目录号:F123600,F123601,F123602和F123603)
    2. 冰箱和冰柜
    3. 生长室(16小时光照/ 8小时黑暗,70-120微米-2秒秒-1,22℃)。
    4. 50毫升离心管(IWAKI)
    5. 大容量离心机(TOMY SEIKO,型号:Suprema 23)
    6. 摇摆转子(TOMY SEIKO,型号:TS-33N和B433)
    7. 摇摆桶(TOMY SEIKO,型号:3350-G01P)

  3. 叶绿体分馏
    1. 移液器(Gilson,型号:P20,P200,P1000和P5000,目录号:F123600,F123601,F123602和F123603)
    2. 冰箱和冰柜
    3. 均质机(IUCHI)
    4. Optima TM TL超速离心机(Beckman Coulter)
    5. 角转子(Beckman Coulter,型号:TLA-100.3,目录号:349490)
    6. 3.5毫升聚碳酸酯超速离心管(Beckman Coulter,目录号:349622)
    7. 摆臂转子(Beckman Coulter,型号:TLS-55,目录号:346134)
    8. 2.2ml Ultra-Clear TM离心管(Beckman Coulter,目录号:347356)

  4. 胰蛋白酶处理完整的叶绿体
    1. 移液器(Gilson,型号:P20,P200,P1000和P5000,目录号:F123600,F123601,F123602和F123603)

  5. 碱性提取叶绿体
    1. 移液器(Gilson,型号:P20,P200,P1000和P5000,目录号:F123600,F123601,F123602和F123603)
    2. 角转子(Beckman Coulter,型号:TLA-100.3,目录号:349490)
    3. Optima TM TL超速离心机(Beckman Coulter)
    4. 1.5ml Polyallomer管(Beckman Coulter,产品目录号:357448,355919)

  6. 用于评估蛋白质浓度的斑点印迹分析
    1. 移液器(Gilson,型号:P20,P200,P1000和P5000,目录号:F123600,F123601,F123602和F123603)
    2. Labo振动筛(BIO CRAFT,型号:BC-740)

程序

  1. 载体的构建和拟南芥转化
    为了向叶绿体IEM递送感兴趣的蛋白质,我们必须将至少两个不同的靶向信号融合到其中。一个信号是转运肽,另一个是叶绿体IEM靶向信号(图3)。已知转运肽作为靶向叶绿体基质的靶向信号,并发现靶向叶绿体内部的大多数前体蛋白质中。然而,转运肽似乎不足以将货物蛋白质,在我们的情况下是蓝藻碳酸氢盐转运蛋白,导向叶绿体IEM。作为额外的IEM靶向信号,我们将Cor413im1蛋白质的成熟部分与图3所示的嵌合构建体融合。这部分可以在转基因植物拟南芥中(上原等, ,2016)。另一项研究显示膜蛋白质前导序列可以在瞬时表达系统中充当IEM靶向信号(Rolland等人,2016)。此外,如果没有特异性抗体,则需要添加标签来检测嵌合蛋白。
    已经在别处详细描述了图3所示的转运肽的长度和Cor413im1的成熟部分(Okawa等人,2008和2014)。


    图3.针对碳酸氢根转运蛋白SbtA II特异性靶向叶绿体IEM的构建设计本研究中使用的嵌合SbtAII构建体的示意图。 TP表示转运肽,并作为叶绿体的靶向信号。构建体的Cor413im1部分来源于Cor413im1的成熟部分,可以作为叶绿体IEM的靶向信号。融合构建体的蛋白A结构域含有来自葡萄球菌蛋白A的两个IgG-结合结构域,并用作检测嵌合蛋白的标签。

  2. 拟南芥叶绿体分离
    基于Smith等人(2002)中描述的方法,我们开发了使用更少数量的植物分离足量的完整叶绿体的方法。如果不能获得大量的种子来分离叶绿体(例如,突变体,转基因品系),则该方法是有用的。在整个过程中,除非另有说明,样品应保存在冰上,并且在实验之前应将缓冲液冷冻。
    1. 在0.5x MS平板上播种灭菌的种子(每个平板100个种子,每个隔离四个平板,参见食谱),补充有1%蔗糖。在16小时/ 8小时黑暗循环下,允许拟南芥属植物在生长室中生长,100μE/ m 2•s -1, ,22℃14-18天(图4A)。
    2. 用刀片收获植物的整个地上部分,并立即放在含有10ml消化缓冲液(参见食谱)的直径为90mm的培养皿上。将从两个平板收获的植物放入一个酶消化反应中,以便您需要为四个拟南芥菜板制备两个直径为90mm的培养皿。当所有的组织都被收获后,使用剃刀片快速切碎组织1分钟(图4B)。
      注意:切勿超过1分钟或粉碎组织。
    3. 从培养皿中取出消化缓冲液。加入13.5毫升消化酶溶液(见食谱)到培养皿中,用指尖均匀分布组织(图4C)。将培养皿置于塑料容器中,并在22℃下在生长室中孵育3小时(光强度约为50-100μEm-2 s -1 )。
    4. 在消化过程中,在50ml离心管中准备40%(20ml,上层):85%(7ml,下层)AT Percoll(见食谱)梯级,并保持在冰上。我们对两个拟南芥板使用一个梯度(一个酶消化),所以我们通常为一个分离准备两个梯度。你也可以使用较小的Percoll体积和管(Smith等人,2002年)。
    5. 通过平板轻轻旋转(或温和搅动)30-60秒,收获原生质体,然后通过200微米的锥形滤网,放入一个小漏斗,在冰上的50毫升离心管过滤介质。从一个培养皿收集原生质体到一个50毫升管,你需要准备两个管。将尼龙网中的任何组织转移回培养皿中,并用10ml冰冷的新鲜消化缓冲液洗涤组织。将组织缓冲液混合物过滤到相同的50ml离心管中。将组织再清洗一次或两次,以确保尽可能多的原生质体释放(图4D)。
    6. 在100℃,4℃下将原生质体离心5分钟(图4E)。用吸气器小心取出并丢弃上清液。小心不要打扰原生质球,因为它很松散。
    7. 在5毫升原生质体重悬缓冲液(见食谱)中重悬沉淀,轻轻地旋转沉淀,同时将缓冲液分配到管的侧面。原生质体重新悬浮后,再加入5毫升原生质体重悬缓冲液,轻轻混合(图4F)。在100℃,4℃下将原生质体离心4分钟(图4G)。
    8. 去除上清液,并悬浮在5毫升原生质体破碎缓冲液颗粒(见食谱)。原生质体重新悬浮后,再添加5毫升原生质体破碎缓冲液,轻轻混合(图4H)。立即将重新悬浮的原生质体沉淀转移到原生质体破裂装置的桶中。将装置的末端保持在冰上的50ml离心管上,小心地更换柱塞,轻轻地将悬浮液通过网格层(图4I)。重复此过程一次。
    9. 快速小心地将10毫升破碎的原生质体铺在AT Percoll台阶梯度上(图4J)。在2,500×g,4℃下,在摆动转子中离心10分钟,关闭制动器(图4K)。离心后,梯度上应该有两条可见的绿色条带:在原生质体破碎缓冲液/ 40%Percoll界面的破碎的叶绿体上带,以及在40%/ 85%Percoll界面下的完整叶绿体的下带。通过抽吸去除负载区和破碎的叶绿体的上层带。使用巴斯德吸管收获较低的带,并用50ml离心管(图4L)中的40-45ml HEPES-山梨糖醇缓冲液(见配方),pH8.0稀释完整的叶绿体。来自两个AT Percoll梯度的完整叶绿体应合并成一个管。
    10. 在700℃,4℃离心稀释的完整叶绿体5分钟(图4M)。小心地滗析上清液,丢弃所有过量的缓冲液,不要中断沉淀。重悬沉淀物(200-300μl)HEPES山梨糖醇缓冲液,pH值为8.0(图4N)。
    11. 将5μl叶绿体再悬浮液稀释到995μl80%丙酮中。剧烈混合并在约15,000×g下微离心2分钟以除去蛋白质沉淀。用80%的丙酮空白测量上清液的A 652。通过使用以下等式(Smith等人,2002)计算叶绿素浓度(mg / ml):
      (A <652/36)×200
      以弥补稀释因素。使用HEPES-山梨糖醇缓冲液将叶绿素浓度调节至1mg叶绿素/ ml。制备的叶绿体应立即用于程序C和D,而用于程序E的叶绿体可以在-80℃下保存。


      图4.拟南芥叶绿体分离概述(程序B)

  3. 叶绿体分馏
    1. 将步骤B11中制备的0.5毫升1毫克叶绿素/毫升叶绿体在700℃×4℃下离心5分钟。去除上清液并通过移液测量上清液的体积。在含有0.6M蔗糖的TE / DTT缓冲液(见配方)中将叶绿体沉淀重悬浮至浓度为1mg / ml叶绿素(图5A)。让我们站在冰上10分钟。在-20°C冻结叶绿体悬液1-2 h。如果不立即进入分馏阶段,可以将冷冻的叶绿体悬液保持在-80°C直到使用。
    2. 解冻悬浮液,用3倍体积的TE / DTT缓冲液稀释。为了收集叶绿体总蛋白,在加入TE / DTT之前,将5μl叶绿体悬浮液加入到1.5ml微管中。在Dounce(或Potter)匀浆器中用紧杵匀浆20次(图5B)。将悬浮液转移到3.5ml聚碳酸酯超速离心管中。在固定角度的转子中,在40,000×gg,4℃下超速离心裂解的叶绿体1小时(图5C)。
    3. 使用移液管移除含有基质成分的褐色上清液并储存在-80℃(图5D)。通过移液(图5D)和匀化(图5E,在Dounce匀浆器中20次冲击),将含有0.2M蔗糖的TE / DTT缓冲液中的膜沉淀重悬浮至浓度为1mg叶绿素/ ml。
    4. 在2.2ml多聚体离心管中建立由1ml 1M蔗糖和0.7ml 0.46M蔗糖组成的蔗糖梯度梯度。将膜悬浮液(0.2M蔗糖)铺在顶部(图5F和图6A)。在每个梯度上应用0.5毫升膜悬浮液。使用低加速和减速速率在摇摆转子转子中以270,000×g,4℃超速离心样品1.5小时(图5G)。从梯度的每一步收集界面(图5H和图6B)。上界面(0.2:0.46 M蔗糖)含有残余的基质和plagoglobules。中间界面(0.46:1M蔗糖)高度富集信封膜。类囊体膜在管的底部形成紧密的小球。
    5. 通过加入100%三氯乙酸(TCA)使终浓度为10%和tRNA达到10μg/ ml的终浓度来沉淀基质蛋白质(在步骤C2中收集)。在冰上孵育60分钟或更长时间。通过在〜20,000×g,4℃下离心30分钟收集TCA沉淀物。用1ml的80%丙酮洗涤沉淀并在〜20,000×g,4℃下离心15分钟。直接在200μlSDS样品缓冲液中重悬沉淀(见食谱)。
    6. 沉淀总叶绿体蛋白质(步骤C2中收集5μl),加入1ml 80%丙酮并涡旋。将管保持在冰上30分钟,并在〜20,000×g,4℃下离心30分钟。直接在50μlSDS样品缓冲液中重悬沉淀。
    7. 为了收集包膜,用3至5体积的TE / DTT缓冲液稀释0.46:1M蔗糖界面部分,并在270,000 emg,4℃图5J)。用移液管取出上清液并丢弃。将沉淀重悬于30μlSDS-样品缓冲液中进行分析(图5K)。
    8. 将类囊体团粒悬浮在0.3ml SDS-样品缓冲液中(图5H)。
    9. 在每个部分中的蛋白质定量后,通过SDS-PAGE分析总叶绿体(3μg),基质(3μg),包膜(1μg)和类囊体(1.5μg)部分。


      图6.用于分离包膜和类囊体膜的蔗糖密度梯度超速离心的示意图(步骤C4)。该实验使用2.2ml Ultra-Clear TM离心管。 A和B分别表示超速离心前后的蔗糖密度梯度。

  4. 胰蛋白酶处理完整的叶绿体
    胰蛋白酶能渗透完整的叶绿体的外膜膜(OEM)而不是内膜膜(IEM)(Jackson等人,1998; Inaba等人的 ,2003)。因此,定位于OEM的蛋白质如Toc75对胰蛋白酶处理敏感。这种方法使我们能够确定一个感兴趣的蛋白质是否定位于叶绿体的OEM或IEM。
    1. 用125μlHEPES-山梨糖醇缓冲液稀释25μl1mg叶绿素/ ml完整叶绿体(步骤B11)。
    2. 加150μL2x胰蛋白酶缓冲液(见食谱),轻轻混合,并在冰上孵育30-40分钟。
    3. 停止反应加入300μL的2个停止缓冲液(见食谱)。颠倒管子数次,小心混合,在冰上孵育10分钟。在冰上孵育后,通过1×40%Percoll(参见配方)在2,500×g,4℃下离心5分钟来分离叶绿体。
    4. 小心吸出上清液和1×40%Percoll,含有一些破碎的叶绿体。
    5. 在500μl的1x HEPES-山梨糖醇缓冲液中重悬叶绿体沉淀(参见食谱)。在2,500×g,4℃下离心叶绿体2分钟。去除上清液,并悬浮在50μL的SDS - c omplete TM缓冲液(见食谱)颗粒。

  5. 碱性抽提叶绿体
    为了研究感兴趣的蛋白质是否整合到包膜中,可以对叶绿体进行碱提取。如果蛋白质被整合到包膜中,则应在碱提取和随后的超速离心后将其回收在沉淀部分中。在外周相关的IEM蛋白如Tic22在提取过程中从IEM释放(Kouranov等,1998)。
    1. 在1.5ml多聚体共聚物试管中,将25μl1mg叶绿素/ ml叶绿体(步骤B11)稀释到1ml 0.2M Na 2 CO 3,pH 12中,在冰上10分钟。
    2. 在一个固定角度的转子中以100000×g,4℃超速离心该混合物15分钟。小心地收集含有基质的上清液并转移到新鲜的试管中。用80μlSDS-样品缓冲液重悬膜沉淀。彻底溶解颗粒。
    3. 通过添加100%(w / v)TCA至终浓度10%(w / v)和tRNA至终浓度10μg/ ml来沉淀碱性上清液。在冰上孵育30至60分钟。
    4. 通过在〜20,000×g,4℃下离心30分钟收集TCA沉淀物。用1ml的80%丙酮洗涤沉淀并在〜20,000×g,4℃下离心15分钟。
    5. 直接重悬沉淀80μLSDS样品缓冲液。
      完全溶解颗粒
  6. 用于评估蛋白质浓度的斑点印迹分析 程序C和D的样品浓度估计如下进行:
    1. 用Whatman 3M 3MM色谱纸(3MM纸)裁下一个正方形,并放在一张干净的纸巾上。

    2. 标记BSA标准的浓度,并用铅笔在3MM纸上测试每个样本(见图7)。
    3. 用SDS-样品缓冲液稀释2.0mg / ml牛血清白蛋白(BSA),制成BSA标准品(0.2,0.4,0.6,0.8,1.0,1.2和1.5mg / ml)。用3μlSDS-样品缓冲液稀释3μl待测样品。如果蛋白质浓度预计很高,请按图7所示进行连续稀释。
    4. 轻轻地点2微升BSA标准和每个样品到移液器的3MM纸上。
    5. 将3MM纸在室温下干燥至少20分钟。
    6. 在考马斯蓝染色的摇床上染色30分钟。
    7. 尽可能收集考马斯蓝染色剂(可重复使用)。
    8. 用考马斯亮点解决方案在振荡器上脱纸。每20分钟更换一次destain解决方案。直到背景清晰。

    9. 在纸巾上擦干3MM纸

    10. 通过比较印迹颜色的强度和BSA标准来估计每个样本的浓度

数据分析

蛋白质浓度的测定和SDS-PAGE

  1. 比较每种信号与BSA标准品的强度并确定蛋白质的浓度(图7:例如),1/2稀释的Cp斑点的信号强度几乎等于0.6mg / ml BSA标准点)。
  2. 根据每个级分中蛋白质的量化准备样品。样品体积可以使用SDS-样品缓冲液进行调整。
    注意:在大多数情况下,每个部分的一部分足以检测感兴趣的蛋白质。 Cp:Str:Env:Thy(程序C中制备)的蛋白质比率应始终为3:3:1:1.5。


    图7.蛋白质浓度的斑点印迹定量。上部分表示BSA标准品(STD)的染色。将分离的叶绿体(Cp)在程序C中分离成基质(Str),包膜(Env)和类囊体膜(Thy)级分。每个样品用SDS-样品缓冲液稀释2,4,8,16,32次。

  3. 通过SDS-PAGE分离每个级分中回收的蛋白质(程序C,D和E),并通过Western印迹分析。
  4. 研究嵌合蛋白在叶绿体内的定位。作为一个例子,我们使用了SbtAII。使用诸如LSU(基质),Tic110(包膜)和LHCP(类囊体膜)的标记蛋白来确认每个级分的纯度。 SbtAII融合蛋白定位于叶绿体中(图8,泳道Cp)。此外,发现SbtAII融合蛋白高度富集包膜部分(图8,泳道Env)。


    图8.嵌合蛋白SbtAII在拟南芥叶绿体中的定位(程序C)。分离出的叶绿体(Cp)分为基质(Str),包膜(Env)和类囊体膜(Thy)。在这些分析中使用的Cp:Str:Env:Thy的蛋白质比率始终是3:3:1:1.5。
    用抗蛋白A,LSU,Tic110和LHCP的抗体进行免疫印迹
  5. 研究SbtAII是否是外膜或内膜膜蛋白。胰蛋白酶渗透完整的叶绿体的外膜膜,而不是内膜膜。正如所料,OEM蛋白Toc75被胰蛋白酶消化(图9,Toc75)。相反,嵌合SbtAII(图9,蛋白A)和IEM蛋白Tic110(图9,Tic110)对胰蛋白酶具有抗性,表明嵌合蛋白定位于叶绿体的IEM。


    图9.嵌合SbtA蛋白在叶绿体中的胰蛋白酶敏感性(程序D)包括外膜蛋白Toc75的蛋白酶敏感性作为阳性对照。内包膜蛋白Tic110的蛋白酶抗性也被列为对照。

  6. 研究SbtAII是否被整合到IEM中,或者是与IEM外围相关联的。 Tic110是一个完整的IEM蛋白质,并显示为阳性对照。 SbtAII对碱性提取具有抗性(图10),表明SbtAII是叶绿体IEM中完整的膜蛋白。


    图10.嵌合SbtA II蛋白在叶绿体的可溶性和膜级分中的定位(程序E)。用碱性溶液处理的叶绿体分离成不溶性(P)和可溶性(S)级分。包括不溶性蛋白Tic110和可溶性蛋白LSU作为对照。

食谱

  1. 0.5倍Murashige和Skoog培养基(MS培养基)(每片60ml)
    0.23克Murashige和Skoog植物盐混合物(最后0.5倍)
    1克蔗糖(最终1%(w / v)) 0.05克MES(最终2.3mM)
    ddH 2 O至100ml,使用1N KOH调节pH至5.7-5.8 调整后,加入0.5克琼脂(最后0.5%)和高压灭菌器
  2. 消化缓冲液(4°C储存2周)
    1.56克MES(最终20毫摩尔)
    29.13克山梨醇(最终400毫摩尔)
    200μl1M CaCl 2(最终0.5mM)
    ddH 2 O至400ml,使用1N KOH调节pH至5.2
  3. 消化酶缓冲液

    溶解30克消化缓冲液中的0.6克纤维素酶和0.12克macerozyme 在2,000×g离心10分钟以沉淀不溶性物质
    立即使用上清液
  4. 40%(v / v)AT Percoll(-20°C储存1个月,使用前立即解冻)
    40毫升Percoll(最后40%(V / V))
    50毫升梯度缓冲液(最后50%(V / V))
    10毫升ddH 2 O
  5. 85%(v / v)AT Percoll(-20°C储存1个月,使用前立即解冻)
    42.5毫升Percoll(最终85%(V / V))
    2.5ml 1M HEPES-KOH,pH7.5(最终50mM)
    3.01克山梨醇(最终330毫米)
    加入ddH 2 O至50毫升
  6. 梯度缓冲液(在4°C下保存2周)
    10ml 1M HEPES-KOH,pH 7.5(最终100mM)
    12.04克山梨醇(最终660毫摩尔)
    200μl1M MgCl 2(最终2mM)
    200μl1M MnCl 2(最终2mM)
    800μl0.5M EDTA(最终4mM)
    0.2克BSA(最后0.2%(w / v))(使用前立即添加)
    ddH 2 O至100毫升
  7. 原生质体再悬浮缓冲液(4°C下保存2周)
    0.39克MES(最终20毫米)
    7.29克山梨醇(最终400毫摩尔)
    50μl1M CaCl 2(最终0.5mM)
    ddH2O至100ml,使用1N KOH调节pH至6.0
  8. 原生质体破碎缓冲液(4°C下保存2周)
    0.36克Tricine(最终20 mM)
    5.47克山梨醇(最终300毫摩尔)
    1ml 0.5M EDTA(最终5mM)
    0.19 g EGTA(最终5 mM)
    0.084g NaHCO 3(最终10mM)
    0.1克BSA(最终0.1%(w / v))(在使用前立即添加)
    ddH2O至100ml,使用1N KOH调节pH至8.4
  9. HEPES-山梨糖醇缓冲液,pH8.0(在4℃下储存2周)
    15ml 1M HEPES-KOH,pH8.0(最终50mM)
    18.06克山梨糖醇(最终330毫摩尔)
    ddH 2 O至300毫升
  10. 含有1,0.6,0.46,0.2和0M蔗糖的TE / DTT缓冲液(在4℃下储存2周)
    0.25 ml 1 M Tricine-KOH,pH 7.5(最终50 mM)
    20μl0.5M EDTA(最终2mM)
    5μl1 M DTT(最终1 mM)(使用前立即添加)
    1.712,1.046,0.787,0.343g蔗糖(最终的1,0.6,0.46,0.2和0μM)
    ddH2O至5ml
  11. SDS-样品缓冲液(-20℃保存待用)
    0.35毫升1M Tris碱(最终350毫摩尔)
    0.25ml 20%(w / v)SDS(最后5%(w / v)) 80μl1 M DTT(最终80 mM)
    0.15ml 50%(v / v)甘油(最终7.5%(v / v)) 40μl饱和溴酚蓝(最后1%(v / v))
    0.13ml ddH 2 O
  12. 2x胰蛋白酶缓冲液
    8μl1M MgCl 2(最终8mM)
    0.2μl1M CaCl 2(最终0.2mM)
    HEPES山梨糖醇缓冲液1毫升

    在300μl2x胰蛋白酶缓冲液中加入6.6μl20mg / ml胰蛋白酶
  13. 2个停止缓冲区
    添加2微升50毫克/毫升TLCK
    2μl2 mg / ml抑肽酶
    10μl200 mM PMSF
    20μl10 mg / ml胰蛋白酶抑制剂
    20μl0.5M EDTA
    在1ml HEPES-山梨糖醇缓冲液中
  14. 1倍40%Percoll
    加入1微升50毫克/毫升TLCK
    1μl2 mg / ml抑肽酶
    5μl200mM PMSF
    10μl10 mg / ml胰蛋白酶抑制剂
    10μl0.5M EDTA
    在1毫升40%Percoll
  15. 1倍HEPES山梨醇缓冲液
    加入1微升50毫克/毫升TLCK
    1μl2 mg / ml抑肽酶
    5μl200mM PMSF
    10μl10 mg / ml胰蛋白酶抑制剂
    10μl0.5M EDTA
    在1ml HEPES-山梨糖醇缓冲液中
  16. SDS-cOmplete 缓冲区
    10μl0.5M EDTA(最终10mM)
    20μl25x cOmplete TM EDTA不含EDTA(最终1x)
    470μlSDS-样品缓冲液
  17. 考马斯蓝染色(在室温下储存)
    0.125克考马斯蓝R250(终浓度0.125%(w / v))
    50ml甲醇(最终50%(v / v))) 10ml乙酸(最终10%(v / v))) ddH 2 O至100毫升
  18. 考马斯亮丽溶液(室温储存)
    45毫升甲醇(最后45%(v / v)) 10ml乙酸(最终10%(v / v))) 45毫升ddH 2 O

致谢

这个协议是从以前发表的作品(上原等人,2016年)修改的。这项工作得到了JSPS KAKENHI基金号码17J06506(至S.U.),17K07762(至Y.I.I)和15K07843(至T.I.)的支持。作者声明不存在利益冲突或利益冲突。

参考

  1. Inaba,T.,Li,M.,Alvarez-Huerta,M.,Kessler,F.and Schnell,D.J。(2003)。 atTic110可以作为协调蛋白进入叶绿体的基质事件的支架。 J Biol Chem 278(40):38617-38627。
  2. Jackson,D. T.,Froehlich,J.E。和Keegstra,K。(1998)。 叶绿体蛋白质易位装置的内包膜组分Tic110的亲水区面向基质隔离室。 J Biol Chem 273(26):16583-16588。
  3. Kouranov,A.,Chen,X.,Fuks,B。和Schnell,D.J。(1998)。 Tic20和Tic22是叶绿体内膜蛋白进口仪器的新成分。
  4. Okawa,K.,Inoue,H.,Adachi,F.,Nakayama,K.,Ito-Inaba,Y.,Schnell,D.J.,Uehara,S。和Inaba,T.(2014)。 体内定位多叶膜蛋白至叶绿体内膜包括多个跨膜片段。 / a> J Exp Bot 65(18):5257-5265。
  5. Okawa,K.,Nakayama,K.,Kakizaki,T.,Yamashita,T。和Inaba,T。(2008)。 将cor413im蛋白鉴定为叶绿体内包膜的新组分。 Plant Cell Environ 31(10):1470-1483。
  6. Rolland,V.,Badger,M.R。和Price,G.D。(2016)。 将蓝细菌碳酸氢盐转运蛋白BicA和SbtA重定向到叶绿体包膜:可溶性和膜状货物需要不同的叶绿体靶向在植物中的信号。植物科学前线7:185.
  7. Smith,M.D。,Schnell,D.J.,Fitzpatrick,L。和Keegstra,K。(2003)。 叶绿体蛋白进口的体外分析 > Curr Protoc Cell Biol Chapter 11:Unit11 16.
  8. Uehara,S.,Adachi,F.,Ito-Inaba,Y.和Inaba,T。(2016)。 蓝细菌碳酸氢盐转运蛋白在拟南芥叶绿体内膜包膜上的特异性和高效靶向性/ 。 Front Plant Sci 7:16.
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引用:Uehara, S., Ito-Inaba, Y. and Inaba, T. (2018). Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana. Bio-protocol 8(3): e2723. DOI: 10.21769/BioProtoc.2723.
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