拟南芥叶绿体内嵌合转运蛋白的定位研究

Susumu Uehara; Yasuko Ito-Inaba; Takehito Inaba

doi:10.21769/BioProtoc.2723

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Peer-reviewed

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

SU Susumu Uehara

YI Yasuko Ito-Inaba

TI Takehito Inaba email

发布: 2018年02月05日第8卷第3期 DOI: 10.21769/BioProtoc.2723 浏览次数: 10003

评审: Dennis NürnbergSam-Geun KongAnonymous reviewer(s)

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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

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
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)
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)
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. N_α-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. cOmplete^TM, 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-cOmplete^TM buffer (see Recipes)
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)
Dot blot assay for estimation of protein concentration
1. Pipette tips (20 μl, 200 μl, 1,000 μl and 5 ml tips)
2. Whatman^TM 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)
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

Construction of vectors and Arabidopsis transformation
1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )
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 )
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. Optima^TMTL 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-Clear^TM centrifuge tubes (Beckman Coulter, catalog number: 347356 )
Trypsin treatment of intact chloroplasts
1. Pipettes (Gilson, models: P20, P200, P1000, and P5000, catalog numbers: F123600 , F123601 , F123602 , and F123603 )
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. Optima^TM TL Ultracentrifuge (Beckman Coulter)
4. 1.5 ml Polyallomer tubes (Beckman Coulter, catalog numbers: 357448 , 355919 )
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 )

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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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