Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

1. Initial Materials

1. Soak approximately 55 g of peas for 3 hours in 400 mL of distilled water, and then sow in a plastic tray (35 cm x 20 cm x 6 cm) in soil covered with thin layer of vermiculite.
2. Grow the tray of peas at 20 °C under 12/12 h light/dark (50 µE/m²s) cycle for 9 to 15 days.
3. Prepare protein substrate according to a preferred method.
   Note: We have prepared protein substrates using a variety of methods, including 1) in vitro transcription from purified plasmids followed by translation using wheat germ extracts or rabbit reticulocyte lysates in the presence of [³H]-leucine or [³⁵S]-methionine, 2) in vitro translation using a coupled transcription/translation kit such as the TnT kits from Promega, with radiolabeling as above, and 3) purification of overexpressed protein in E. coli. Radioactive in vitro translation products are generally quenched with 50 mM cold amino acid prior to use in transport reactions. Each of these methods typically requires some optimization for each new protein substrate. For an example of a coupled in vitro system, see Ling and Jarvis (2016)⁹.

2. Chloroplast Isolation and Quantification

Note: The first step in preparation of thylakoids is the isolation of intact chloroplasts¹⁰. All materials should be kept cold during preparation. Resuspension of chloroplasts should be handled gently, as breakage at this step can severely limit subsequent thylakoid yield.

1. Prepare following solutions beforehand.
   1. 2x Grinding Buffer (2xGB): 100 mM K-HEPES pH 7.3, 660 mM sorbitol, 2 mM MgCl₂, 2 mM MnCl₂, 4 mM EDTA, 0.2% BSA.
   2. 2x Import Buffer (2xIB): 100 mM K-Tricine or K-HEPES pH 8.0, 660 mM sorbitol, 8 mM MgCl₂.
      Note: Concentrations of MgCl₂ ranging from 3 mM to 10 mM have been used without observable differences.
2. Prepare a continuous density gradient by centrifuging a mixture of 15 mL of Percoll and 15 mL of 2xGB at 30,900 g in a fixed angle rotor for 30 min at 4 °C with the brake off.
3. Harvest approximately 25 g of 9-15 day old peas and homogenize in 95 mL of 1xGB. Either a blender with sharpened blades or a Polytron have been used successfully. Filter this mixture through at least 2 layers of Miracloth in a funnel.
   Note: Limiting the amount of stem tissue is not necessary but can make the homogenization process easier, thereby reducing chloroplast breakage that can occur with prolonged blending.
4. Pellet at 3,000 g in a swinging bucket rotor for 5 min at 4 °C, and gently resuspend in 1 mL of 1xGB. A paintbrush may be the most gentle resuspension technique.
5. Carefully layer the green suspension on top of the Percoll gradient and centrifuge at 8,000 g in a swinging bucket rotor for 10 min at 4 °C with the brake off.
6. Carefully harvest the lower green band containing intact chloroplasts into a fresh tube. Spin the recovered intact chloroplasts at 1,800 g in a swinging bucket rotor for 5 min at 4 °C, discard the supernatant and gently resuspend the pellet in 1xIB. Repeat this wash step once more, resuspending in 30 mL of 1xIB each time, with the final resuspension in 1 mL of 1xIB.
7. To quantify chlorophyll (Chl) content, mix 10 µL of fully resuspended chloroplasts with 5 mL of 80% acetone and filter through Whatman 1 filter paper (nominal thickness 180 µm). Record the absorbance at 720 nm, 663 nm, and 645 nm in a spectrophotometer and calculate the Chl concentration using the following formula¹¹:
   [Chl] mg/mL = [40.2*(A₆₆₃ – A₇₂₀) + 101.4*(A₆₄₅ – A₇₂₀)]*0.1
8. Adjust chloroplasts to 1 mg/mL Chl equivalents with 1xIB.

3. Isolation of Thylakoids

Note: Thylakoids are prepared by hypotonic lysis of intact chloroplasts. This is achieved by exposing the chloroplasts to a hypotonic buffer lacking sorbitol. Isolated thylakoids can be used for assaying any of the translocation pathways, but stromal extract (SE) must also be isolated during this preparation if either cpSec1 or cpSRP pathways are to be investigated.

1. Prepare ahead of time the following solutions.
   1. Hypotonic Lysis Buffer: 50 mM K-Tricine or K-HEPES pH 8.0, 8 mM MgCl₂.
   2. 2x Crude Stroma Buffer (for concentration of SE): 100 mM K-HEPES pH 8.0, 660 mM sorbitol, 8 mM MgCl₂.
   3. Laemmli Sample Buffer (for SDS-PAGE, supplemented with EDTA): 125 mM Tris pH 6.8, 10% SDS, 20% glycerol, 0.05 mg/mL bromophenol blue, 10% β-mercaptoethanol, 10 mM EDTA.
2. If SE recovery is not necessary, centrifuge the intact chloroplasts at 1,000 g in a swinging bucket rotor for 6 min at 4 °C.
   1. Discard the supernatant and resuspend to 1 mg/mL in Hypotonic Lysis Buffer. Incubate on ice in the dark for 10 min, with occasional mixing.
   2. Recover thylakoids by centrifuging at 3,200 g in swinging bucket rotor for 8 min at 4 °C. Wash the thylakoids twice, resuspending with 1 to 2 mL of lysis buffer each time. Resuspend with 1 mL of 1xIB and requantify Chl as above in the chloroplast isolation protocol.

4. Stromal Extract Recovery and Concentration

1. If SE recovery is necessary, divide the intact chloroplasts into 600 µL aliquots in 1.5 mL microcentrifuge tubes and centrifuge each tube at 1,000 g in swinging bucket rotor for 6 min at 4 °C.
   Note: This volume is convenient when using 1.5 mL microcentrifuge tubes, which helps to prevent disturbance of the eventual pellet for residual membranes.
   1. Resuspend to 1 mg/mL Chl in Hypotonic Lysis Buffer and incubate on ice in the dark for 10 min, as in step 3.2.1.
   2. Centrifuge each tube at 3,200 x g in swinging bucket rotor for 8 min at 4 °C and transfer the light green or yellow supernatant following chloroplast lysis to a new microcentrifuge tube with an equal volume of 2x Crude Stroma Buffer.
   3. Wash thylakoids with 2 volumes of lysis buffer (approximating 0.5 mg/mL) and collect by centrifugation at 3,200 g in swinging bucket rotor for 8 min at 4 °C. Resuspend to 1 mg/mL Chl in 1xIB on ice.
   4. Centrifuge crude stroma at 42,000 g in a fixed angle rotor for 30 min at 4 °C to remove residual membranes. Immunoblotting for outer and inner envelope proteins can be used to verify the purity of the supernatant.
   5. Collect 95% of the volume from each tube without disturbing the small yellow-green pellet. Note the volume taken from each tube.
   6. To prepare the concentrated stromal extract (SE), pool the collected supernatants and concentrate five-fold using a 4 mL 30 kDa MWCO concentrator.
      Note: SE prepared in this manner is equivalent to stroma derived from chloroplasts at 2.5 mg/mL Chl. If necessary, SE can be concentrated ten-fold to 5 mg/mL Chl equivalents. SE will be golden in color and viscous. In the lab’s Legend RT centrifuge with swinging bucket rotor, this requires about 10 min per mL concentrated.

5. Transport through the cpTat Pathway

Note: Unlike the cpSec1 or cpSRP, the cpTat pathway does not require soluble components or exogenously added energy sources; only the light-driven proton motive force is necessary³. Therefore, only isolated thylakoids and substrate protein are required for the assay. Typical substrates are intermediate forms of the 17 kDa (as seen in Figure 1) and 23 kDa subunits of the oxygen evolving complex, iOE17 and iOE23, respectively, but precursor forms, prOE17 and prOE23, can also be successfully transported. Precursor forms have the entire bipartite N-terminal targeting sequence, while intermediate forms have only the thylakoid targeting sequence.

1. Prepare cpTat transport mix with 0.33 mg/mL Chl thylakoids, 5 mM ATP from a stock prepared in 1xIB, and 8 mM dithiothreitol (DTT) from a stock prepared in 1xIB, on ice. ATP is not strictly required for this reaction if conducted under illumination.
2. Initiate transport by introducing 1:30 volume by volume substrate protein in the transport mix. If substrate protein is not prepared in 1xIB, prepare a 1:1 dilution with 2xIB first, then use 1:30  volume by volume of the diluted substrate in the transport mix.
   Note: Concentrations of substrate will vary based on the preparation method. Typically, in vitro preparations will allow for nanomolar to micromolar amounts, while overexpression will allow for micromolar to millimolar amounts. Detection methods must also be considered, as autoradiography and fluorography are more sensitive than immunoblotting.
3. Illuminate the suspension in 80 – 100 µE/m²s of photosynthetically active radiation (PAR) for 10 min at room temperature. If transport kinetics are required, pre-equilibrate both thylakoids and reaction mix to room temperature and illuminate for up to 30 min.
4. Stop the transport reaction with an 8-fold dilution of ice-cold 1xIB and recover thylakoids by centrifugation at 3,200 g in microcentrifuge for 5 min at 4 °C.
5. Remove residual substrate that has not been transported, resuspend the thylakoid pellet in 120 µL of 1xIB and digest external protein by addition of 6 µL of 2 mg/mL thermolysin and 10 mM CaCl₂ in 1xIB.
6. Incubate the protease reaction on ice for 40 min, and then quench the protease by doubling the volume with 25 mM EDTA prepared in 1xIB.
7. Recover thylakoids by centrifuging at 3,200 g in a microcentrifuge for 5 min at 4 °C. Wash recovered thylakoids in 120 μL of 5 mM EDTA in 1xIB and transfer to a new tube.
8. Centrifuge at 3,200 g in a microcentrifuge for 5 min at 4 °C. Resuspend in an appropriate volume of Laemmli Sample Buffer supplemented with 10 mM EDTA. A volume of 40 µL is usually enough to detect substrate on an SDS-PAGE gel and conserves sample for repeat gels when necessary.
9. Place samples in a boiling water bath for 10 min and analyze by SDS-PAGE. Autoradiography, fluorography, or Western blotting of non-native or epitope-tagged proteins have been successfully used for detection of transported proteins.

6. Transport through the cpSec1 Pathway

Note: Transport through the cpSec1 translocon requires the stromal protein cpSecA1^12,13, which can be procured via overexpression in E. coli^14,15 or recovered by concentrating stroma during thylakoid isolation. A typical substrate is the 33 kDa subunit of the oxygen evolving complex (prOE33), as seen in Figure 2.

1. Prepare the cpSec1 transport mix with 0.33 mg/mL Chl thylakoids, 0.83 mg/mL Chl equivalent SE or 1.5 μg of recombinant cpSecA1, and 5 mM ATP on ice, and incubate for 10 min. A typical transport reaction mixture here is 72 μL, up from 60 μL due to the SE addition.
   1. If recombinant cpSecA1 will be used instead of SE, adjust purified cpSecA1 with an equal volume of 2xIB, then dilute to a convenient concentration to add to the transport reactions (e.g., 0.75 µg/µL).
      Note: For long term storage, cpSecA1 is stored on ice in a controlled, refrigerated room after purification as freezing abolishes activity.
2. Initiate transport by introducing 1:10 volume by volume substrate protein to the transport mix. If substrate protein is not prepared in 1xIB, prepare a 1:1 dilution with 2xIB and add 1:10 volume by volume of the diluted protein to the transport mix.
3. Incubate the reaction at room temperature for 10 min under 80 – 100 µE/m²s PAR. Again, longer times may be necessary for kinetics or certain substrates.
4. After light treatment, dilute 8-fold with ice-cold 1xIB and centrifuge reactions at 3200 g in microcentrifuge for 5 min at 4 °C.
5. Treat with thermolysin and quench with EDTA as above in Steps 5.5 through 5.7.
6. Centrifuge at 3,200 g in microcentrifuge for 5 min at 4 °C and resuspend the pellet in an appropriate volume of Laemmli Sample Buffer supplemented with 10 mM EDTA. Place in boiling water bath for 10 min prior to loading on SDS-PAGE gel.

7. Insertion through the cpSRP Pathway

Note: The cpSRP-mediated integration of light harvesting complex proteins (LHCP) seen in Figure 3 requires cpSRP54, cpSRP43, and cpFtsY¹⁶. These components are supplied to the transport reaction through concentrated stromal extract, as described for the cpSec1 transport protocol.

1. Prepare the cpSRP transport mix with 0.33 mg/mL Chl thylakoids, 0.83 mg/mL Chl equivalent SE, and 5 mM ATP on ice and incubate for 10 min.
2. Initiate transport by introducing 1:10 volume by volume substrate protein to the transport mix. If substrate protein is not prepared in 1xIB, prepare a 1:1 dilution with 2xIB and add 1:10 volume by volume of the diluted protein to the transport mix.
3. Incubate reaction at room temperature for 10 min in 80 – 100 µE/m²s PAR.
4. After light treatment, dilute 8-fold with ice-cold 1xIB and centrifuge reactions at 3,200 g in microcentrifuge for 5 min at 4 °C. Discard supernatant and resuspend the pellet in 120 µL of 1xIB.
5. Treat with thermolysin as above in sections 5.5 through 5.7. Thermolysin digestion is necessary here to assess successful membrane integration. Wash recovered thylakoids in 120 μL of 5 mM EDTA in 1xIB and transfer to a new tube.
6. Centrifuge at 3,200 g in microcentrifuge for 5 min at 4 °C and resuspend the pellet again in an appropriate volume of 2x Laemmli buffer supplemented with 10 mM EDTA. Place in boiling water bath for 10 min prior to analysis by SDS-PAGE.
   Note: Integration of mature LHCP (mLHCP) is assessed by the appearance of a protease-protected degradation product (mLHCP-D) approximately 1.5-2 kDa smaller than the uncleaved mLHCP¹⁷.