This manuscript was prepared with funding by the Division of Chemical Sciences, Geosciences, and Biosciences, 408 Office of Basic Energy Sciences of the US Department of Energy through Grant DE-SC0017035

1. Initial Materials
<ol>
	<li>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.</li>
	<li>Grow the tray of peas at 20 &#176;C under 12/12 h light/dark (50 &#181;E/m2s) cycle for 9 to 15 days.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Prepare following solutions beforehand.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C with the brake off.</li>
	<li>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.</li>
	<li>Pellet at 3,000 g in a swinging bucket rotor for 5 min at 4 &#176;C, and gently resuspend in 1 mL of 1xGB. A paintbrush may be the most gentle resuspension technique.</li>
	<li>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 &#176;C with the brake off.</li>
	<li>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 &#176;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.</li>
	<li>To quantify chlorophyll (Chl) content, mix 10 &#181;L of fully resuspended chloroplasts with 5 mL of 80% acetone and filter through Whatman 1 filter paper (nominal thickness 180 &#181;m). 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 &#8211; A720) + 101.4*(A645 &#8211; A720)]*0.1</li>
	<li>Adjust chloroplasts to 1 mg/mL Chl equivalents with 1xIB.</li>
</ol>
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.
<ol>
	<li>Prepare ahead of time the following solutions.
	<ol>
		<li>Hypotonic Lysis Buffer: 50 mM K-Tricine or K-HEPES pH 8.0, 8 mM MgCl2.</li>
		<li>2x Crude Stroma Buffer (for concentration of SE): 100 mM K-HEPES pH 8.0, 660 mM sorbitol, 8 mM MgCl2.</li>
		<li>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% &#946;-mercaptoethanol, 10 mM EDTA.</li>
	</ol>
	</li>
	<li>If SE recovery is not necessary, centrifuge the intact chloroplasts at 1,000 g in a swinging bucket rotor for 6 min at 4 &#176;C.
	<ol>
		<li>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.</li>
		<li>Recover thylakoids by centrifuging at 3,200 g in swinging bucket rotor for 8 min at 4 &#176;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.</li>
	</ol>
	</li>
</ol>
4. Stromal Extract Recovery and Concentration
<ol>
	<li>If SE recovery is necessary, divide the intact chloroplasts into 600 &#181;L aliquots in 1.5 mL microcentrifuge tubes and centrifuge each tube at 1,000 g in swinging bucket rotor for 6 min at 4 &#176;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.
	<ol>
		<li>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.</li>
		<li>Centrifuge each tube at 3,200 x g in swinging bucket rotor for 8 min at 4 &#176;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.</li>
		<li>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 &#176;C. Resuspend to 1 mg/mL Chl in 1xIB on ice.</li>
		<li>Centrifuge crude stroma at 42,000 g in a fixed angle rotor for 30 min at 4 &#176;C to remove residual membranes. Immunoblotting for outer and inner envelope proteins can be used to verify the purity of the supernatant.</li>
		<li>Collect 95% of the volume from each tube without disturbing the small yellow-green pellet. Note the volume taken from each tube.</li>
		<li>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&#8217;s Legend RT centrifuge with swinging bucket rotor, this requires about 10 min per mL concentrated.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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&#160; 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.</li>
	<li>Illuminate the suspension in 80 - 100 &#181;E/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.</li>
	<li>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 &#176;C.</li>
	<li>Remove residual substrate that has not been transported, resuspend the thylakoid pellet in 120 &#181;L of 1xIB and digest external protein by addition of 6 &#181;L of 2 mg/mL thermolysin and 10 mM CaCl2 in 1xIB.</li>
	<li>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.</li>
	<li>Recover thylakoids by centrifuging at 3,200 g in a microcentrifuge for 5 min at 4 &#176;C. Wash recovered thylakoids in 120 &#956;L of 5 mM EDTA in 1xIB and transfer to a new tube.</li>
	<li>Centrifuge at 3,200 g in a microcentrifuge for 5 min at 4 &#176;C. Resuspend in an appropriate volume of Laemmli Sample Buffer supplemented with 10 mM EDTA. A volume of 40 &#181;L is usually enough to detect substrate on an SDS-PAGE gel and conserves sample for repeat gels when necessary.</li>
	<li>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.</li>
</ol>
6. Transport through the cpSec1 Pathway
Note: Transport through the cpSec1 translocon requires the stromal protein cpSecA112,13, which can be procured via overexpression in E. coli14,15 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.
<ol>
	<li>Prepare the cpSec1 transport mix with 0.33 mg/mL Chl thylakoids, 0.83 mg/mL Chl equivalent SE or 1.5 &#956;g of recombinant cpSecA1, and 5 mM ATP on ice, and incubate for 10 min. A typical transport reaction mixture here is 72 &#956;L, up from 60 &#956;L due to the SE addition.
	<ol>
		<li>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 &#181;g/&#181;L). 
		Note: For long term storage, cpSecA1 is stored on ice in a controlled, refrigerated room after purification as freezing abolishes activity.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Incubate the reaction at room temperature for 10 min under 80 &#8211; 100 &#181;E/m2s PAR. Again, longer times may be necessary for kinetics or certain substrates.</li>
	<li>After light treatment, dilute 8-fold with ice-cold 1xIB and centrifuge reactions at 3200 g in microcentrifuge for 5 min at 4 &#176;C.</li>
	<li>Treat with thermolysin and quench with EDTA as above in Steps 5.5 through 5.7.</li>
	<li>Centrifuge at 3,200 g in microcentrifuge for 5 min at 4 &#176;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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate reaction at room temperature for 10 min in 80 &#8211; 100 &#181;E/m2s PAR.</li>
	<li>After light treatment, dilute 8-fold with ice-cold 1xIB and centrifuge reactions at 3,200 g in microcentrifuge for 5 min at 4 &#176;C. Discard supernatant and resuspend the pellet in 120 &#181;L of 1xIB.</li>
	<li>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 &#956;L of 5 mM EDTA in 1xIB and transfer to a new tube.</li>
	<li>Centrifuge at 3,200 g in microcentrifuge for 5 min at 4 &#176;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.</li>
</ol>

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The authors have nothing to disclose.

Chloroplast and Thylakoid isolation
Excessive breakage can result in poor chloroplast isolation and thus poor thylakoid yield after separation in the gradient. It is best to homogenize the harvested tissue gently by ensuring that all material is submerged before blending and pulsing in 15 s cycles until fully homogenized. If necessary, use multiple shorter rounds of blending with less tissue in each round.
Refrigerating all materials that come into contact with harvested tissue helps isolated chloroplasts to retain activity up to 2 hours. It is important to keep the chloroplasts on ice in the dark after isolation as well.
It is crucial to include magnesium in buffers contacting isolated thylakoids. Not doing so results in granal destacking and critically affects the generation of proton motive force upon illumination21. Thylakoids have been used in transport reactions up to 2 hours after isolation when kept on ice and in the dark.
Optimizing Transport Reactions
Isolated thylakoids settle rapidly and can result in unequal Chl equivalents between reactions. As such, it is important to mix the thylakoids thoroughly prior to use, especially when setting up a large number of samples.
The Tat Pathway
Transport efficiency through the Tat pathway can be reduced upon excessive washing of the thylakoids since Tha4 (TatA) can be partially extracted from the membrane22. In such cases, it is helpful to reduce thylakoid wash steps prior to the transport reaction. Additionally, longer illumination times can improve detection where substrates are poorly transported under typical conditions.
The cpSec1 Pathway 
When assaying the cpSec1 pathway, the amount of cpSecA1 in concentrated SE should support transport, but that transport may be weak. Efficient transport in isolated thylakoids may benefit from the addition of purified cpSecA1 for certain proteins, though stromal chaperones in SE may also help increase the efficiency of transport15. Further, preparations of cpSecA1 protein should not be frozen prior to usage in transport, as this reduces transport activity. Similarly, frozen SE is less efficacious than freshly prepared extract. As in Tat transport, longer incubation periods in the transport mix can help with difficult substrates.
The cpSRP Pathway
While reconstitution of cpSRP transport using individual components has been performed23, it is convenient to supply cpSRP43, cpSRP54, and cpFtsY with SE. Thermolysin resistance is the most stringent criteria for LHCP integration16,17, but alkaline extraction may be performed as well17. While precursors can often be frozen and stored at -80 &#176;C for many transport reactions, prLHCP prepared by in vitro synthesis should be used for transport immediately after synthesis without freezing.
Characterization of a Novel Substrate&#39;s Targeting Pathway
In cases where the translocation pathway taken by a novel substrate is unknown, it is advisable to first investigate possible signal peptides in silico using the amino acid sequence of the precursor or intermediate form. The cpTat pathway typically requires a specific twin arginine consensus motif in the signal peptide and no ATP for successful transport under PAR3. Unlike the Tat pathway, the cpSec1 pathway requires ATP and the cpSecA1 protein. Failed transport in conditions lacking these components suggests the cpSec1 pathway20. The cpSRP pathway requires the signal recognition particle found in the SE. Failed transport using purified cpSecA1 and ATP, but a lack of twin arginine consensus motif in the signal peptide, suggests the cpSRP pathway23.

Nearly all of the proteinaceous machinery responsible for proper chloroplast function must be translocated from the cytosol1. At the chloroplast envelopes, protein substrates are imported through the translocon of the outer membrane (TOC) and the translocon of the inner membrane (TIC)2. Further targeting to the thylakoid membrane and lumen occurs through the twin arginine translocation (cpTat)3, the secretory (cpSec1)4, the signal recognition particle (cpSRP)5, and the spontaneous insertion pathways6. A method for the high-yield isolation of physiologically active chloroplasts and thylakoid membranes is necessary to measure the energetics and kinetics of a translocation event, to understand the diverse transport mechanisms in each pathway, and to localize a particular protein substrate of interest to any of the six distinct compartments of the chloroplast.
The isolation of membranes from the chloroplast offers better experimental control over environmental factors (such as salt and substrate concentrations, the presence of ATP/GTP, and pH conditions) that affect the measurement of transport energetics and kinetics. This in vitro environment lends itself to the exploration of mechanistic details of translocation for the same reasons. In addition, while predictive software for localization of chloroplast proteins has improved7,8, in vitro transport assays provide a quicker method for confirmation over microscopy-based fluorescent assays that require a genetically encoded fluorescent tag, plant transformation and/or specific antibodies. Here, we present protocols for chloroplast and thylakoid isolations from peas (Pisum sativum), as well as for transport assays optimized for each of the energy-dependent thylakoid translocation pathways.

<table><tbody><tr><td>&lt;em&gt;Pisum sativum&lt;/em&gt; seeds</td><td>Seedway LLC, Hall, NY</td><td>8686 - Little Marvel</td><td></td></tr><tr><td>Miracloth</td><td>Calbiochem, Gibbstown, NJ</td><td>475855-1</td><td></td></tr><tr><td>80% Acetone</td><td>Sigma, Saint Louis, MO</td><td>67-64-1</td><td></td></tr><tr><td>Blender with sharpened blades</td><td>Waring Commercial</td><td>BB155S</td><td></td></tr><tr><td>Polytron 10-35</td><td>Fischer Sci</td><td>13-874-617</td><td></td></tr><tr><td>Percoll</td><td>Sigma, Saint Louis, MO</td><td>GE17-0891-01</td><td></td></tr><tr><td>Beckman J2-MC with JA 20 rotor</td><td>Beckman-Coulter</td><td>8043-30-1180</td><td></td></tr><tr><td>Sorvall RC-5B with HB-4 rotor</td><td>Sorvall</td><td>8327-30-1016</td><td></td></tr><tr><td>100 mM dithiothreitol (DTT) in 1xIB</td><td>Sigma, Saint Louis, MO</td><td>12/3/83</td><td>Can be frozen in aliquots for future use</td></tr><tr><td>200 mM MgATP in 1xIB</td><td>Sigma, Saint Louis, MO</td><td>74804-12-9</td><td>Can be frozen in aliquots for future use</td></tr><tr><td>Thermolysin in 1xIB (2mg/mL)</td><td>Sigma, Saint Louis, MO</td><td>9073-78-3</td><td>Can be frozen in aliquots for future use</td></tr><tr><td>HEPES</td><td>Sigma, Saint Louis, MO</td><td>H3375</td><td></td></tr><tr><td>K-Tricine</td><td>Sigma, Saint Louis, MO</td><td>T0377</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma, Saint Louis, MO</td><td>50-70-4</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma, Saint Louis, MO</td><td>7791-18-6</td><td></td></tr><tr><td>Manganese Chloride</td><td>Sigma, Saint Louis, MO</td><td>13446-34-9</td><td></td></tr><tr><td>EDTA</td><td>Sigma, Saint Louis, MO</td><td>60-00-4</td><td></td></tr><tr><td>BSA</td><td>Sigma, Saint Louis, MO</td><td>9048-46-8</td><td></td></tr><tr><td>Tris</td><td>Sigma, Saint Louis, MO</td><td>77-86-1</td><td></td></tr><tr><td>SDS</td><td>Sigma, Saint Louis, MO</td><td>151-21-3</td><td></td></tr><tr><td>Glycerol</td><td>Sigma, Saint Louis, MO</td><td>56-81-5</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Sigma, Saint Louis, MO</td><td>115-39-9</td><td></td></tr><tr><td>B-Mercaptoethanol</td><td>Sigma, Saint Louis, MO</td><td>60-24-2</td><td></td></tr></tbody></table>

isolation of physiologically active thylakoids and their use in energy-dependent protein transport assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

To gauge amount of substrate successfully transported, it is useful to include one or more &#34;percent input&#34; lanes. For the data presented below, 10% of the final transport reaction without thylakoids was used. This &#34;percent input&#34; also helps to visualize the size of the precursor substrate. The percentage represents a known, defined amount of substrate with which to compare transported substrate against and can be scaled up or down as necessary using initially prepared protein. Additionally, it is advisable to load less than 4 &#181;g of Chl equivalents in a single lane on 0.75 mm polyacrylamide gels to avoid band warping and smearing. All substrates below were prepared and radiolabeled using in vitro translation kits.
Transport of iOE17 through cpTat pathway
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58393/58393fig1.jpg" /> 
Figure 1. Transport of iOE17through the cpTat pathway. Fluorograph of [3H]-iOE17 transport assay and thermolysin treatment of non-transported substrate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58393/58393fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Successful cpTat transport of most Tat substrates can be detected by a size shift upon cleavage of the N-terminal signal peptide3. In substrates where no cleavable signal peptide exists18,19, protease treatment is required to reveal the substrate that crossed the membrane, thereby becoming inaccessible to digestion by the protease. In Figure 1, transport of iOE17 results in a size shift of approximately 2-3 kDa between the introduced substrate and the mature processed form. Non-transported substrate is degraded through protease treatment (lane 4).
Transport of prOE33 through cpSec1 pathway
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58393/58393fig2.jpg" /> 
Figure 2. Transport of prOE33 through cpSec1 pathway. Autoradiograph of [35S]-prOE33 transport assay using SE, purified cpSecA1, or both simultaneously. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58393/58393fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Transport through the cpSec1 pathway (Figure 2) can be evaluated through a size shift in the mature substrate if the substrate contains a cleavable thylakoid targeting signal, as well as protease protection of transported substrate20.
Integration of prLHCP through cpSRP pathway
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58393/58393fig3.jpg" /> 
Figure 3. Integration of prLHCP through cpSRP pathway. Autoradiograph of [35S]-prLHCP and digestion to the product mLHCP-D after insertion into the thylakoid membrane. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58393/58393fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Insertion of prLHCP into the thylakoid membrane via the cpSRP pathway can be evaluated through thermolysin digestion. A size shift of approximately 1.5-2 kDa is indicative of successful membrane insertion as the membrane protects the uncleaved mature protein from complete proteolysis15. In Figure 3, the protease-protected product mLHCP-D is clearly visible.

Watch this Scientific Journal Video about Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays at JoVE.com

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

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

isolation-physiologically-active-thylakoids-their-use-energy

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.8K Views.  University of California - Davis. Deze methode kan helpen bij het beantwoorden van belangrijke vragen met betrekking tot de mechanismen en energetische stoffen van eiwittransport over chloroplast membranen, evenals de locatie van eiwitten binnen plastiden. Het belangrijkste voordeel van deze techniek voor thylakoïde eiwittargeting is dat het volledige controle over de omgeving van de thylakoïde tijdens het transport mogelijk maakt, inclusief temperatuur, pH, ionische omstandigheden en precursorconcentraties. Begin met het w...

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

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and organization of these complexes is highly dynamic on both long and short time scales due to processes that adapt photosynthesis to changing environmental conditions (i.e., non-photochemical quenching, state transitions, and the long-term response). Historically, these processes have been described spectroscopically in terms of changes in chlorophyll fluorescence, and spectroscopy remains a vital method for monitoring photosynthetic parameters. There are a limited number of ways in which the underlying protein complex dynamics can be visualized. Here we describe a fast and simple method for the high-resolution separation and visualization of thylakoid complexes, native green gel electrophoresis. This method is coupled with time-correlated single photon counting for detailed characterization of the chlorophyll fluorescence properties of bands separated on the green gel.

Here we present a protocol to separate solubilized thylakoid complexes by Native Green Gel electrophoresis. Green gel bands are subsequently characterized by Time Correlated Single Photon Counting (TCSPC) and basic steps for data analysis are provided.

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and ...

Environment

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Chemistry

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Biology

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

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

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