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 fro...

<ol>
	<li>Ellis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+protein+synthesis:+principles+and+problems.">Chloroplast protein synthesis: principles and problems.</a> Sub-cellular biochemistry. 9, 237 (1983).</li><li>Li, H. -. m., Chiu, C. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transport+into+chloroplasts.">Protein transport into chloroplasts.</a> Annual review of plant biology. 61, (2010).</li><li>Cline, K., Ettinger, W., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-specific+energy+requirements+for+protein+transport+across+or+into+thylakoid+membranes.+Two+lumenal+proteins+are+transported+in+the+absence+of+ATP.">Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.</a> Journal of Biological Chemistry. 267 (4), 2688-2696 (1992).</li><li>Skalitzky, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastids+contain+a+second+sec+translocase+system+with+essential+functions.">Plastids contain a second sec translocase system with essential functions.</a> Plant physiology. 155 (1), 354-369 (2011).</li><li>Dabney-Smith, C., Storm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastid Biology. , 271-289 (2014).</li><li>Kim, S. J., Jansson, S., Hoffman, N. E., Robinson, C., Mant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+&amp;#34;assisted&amp;#34;+and+&amp;#34;spontaneous&amp;#34;+mechanisms+for+the+insertion+of+polytopic+chlorophyll-binding+proteins+into+the+thylakoid+membrane.">Distinct &amp;#34;assisted&amp;#34; and &amp;#34;spontaneous&amp;#34; mechanisms for the insertion of polytopic chlorophyll-binding proteins into the thylakoid membrane.</a> Journal of Biological Chemistry. 274 (8), 4715-4721 (1999).</li><li>Emanuelsson, O., Nielsen, H., Von Heijne, G. C. h. l. o. r. o. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChloroP,+a+neural+network-based+method+for+predicting+chloroplast+transit+peptides+and+their+cleavage+sites.">ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites.</a> Protein Science. 8 (5), 978-984 (1999).</li><li>Emanuelsson, O., Brunak, S., Von Heijne, G., Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locating+proteins+in+the+cell+using+TargetP,+SignalP+and+related+tools.">Locating proteins in the cell using TargetP, SignalP and related tools.</a> Nature protocols. 2 (4), 953 (2007).</li><li>Ling, Q., Jarvis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+import+into+chloroplasts+isolated+from+stressed+plants.">Analysis of protein import into chloroplasts isolated from stressed plants.</a> Journal of Visualized Experiments. (117), e54717 (2016).</li><li>Lo, S. M., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Photosynthesis Research Protocols. , 139-157 (2011).</li><li>Vernon, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+determination+of+chlorophylls+and+pheophytins+in+plant+extracts.">Spectrophotometric determination of chlorophylls and pheophytins in plant extracts.</a> Analytical Chemistry. 32 (9), 1144-1150 (1960).</li><li>Knott, T. G., Robinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secA+inhibitor,+azide,+reversibly+blocks+the+translocation+of+a+subset+of+proteins+across+the+chloroplast+thylakoid+membrane.">The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane.</a> Journal of Biological Chemistry. 269 (11), 7843-7846 (1994).</li><li>Yuan, J., Henry, R., McCaffery, M., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SecA+homolog+in+protein+transport+within+chloroplasts:+evidence+for+endosymbiont-derived+sorting.">SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting.</a> Science. 266 (5186), 796-798 (1994).</li><li>Nohara, T., Nakai, M., Goto, A., Endo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+the+cDNA+for+pea+chloroplast+SecA+Evolutionary+conservation+of+the+bacterial-type+SecA-dependent+protein+transport+within+chloroplasts.">Isolation and characterization of the cDNA for pea chloroplast SecA Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts.</a> FEBS letters. 364 (3), 305-308 (1995).</li><li>Endow, J. K., Singhal, R., Fernandez, D. E., Inoue, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone-assisted+post-translational+transport+of+plastidic+type+I+signal+peptidase+1.">Chaperone-assisted post-translational transport of plastidic type I signal peptidase 1.</a> Journal of Biological Chemistry. 290 (48), 28778-28791 (2015).</li><li>Luirink, J., Sinning, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRP-mediated+protein+targeting:+structure+and+function+revisited.">SRP-mediated protein targeting: structure and function revisited.</a> Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1694 (1-3), 17-35 (2004).</li><li>Yuan, J., Henry, R., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+factor+plays+an+essential+role+in+protein+integration+into+thylakoids+that+cannot+be+replaced+by+unfolding+or+by+heat+shock+protein.">Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein.</a> Hsp70. Proceedings of the National Academy of Sciences. 90 (18), 8552-8556 (1993).</li><li>Tjalsma, H., van Dijl, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics-based+consensus+prediction+of+protein+retention+in+a+bacterial+membrane.">Proteomics-based consensus prediction of protein retention in a bacterial membrane.</a> Proteomics. 5 (17), 4472-4482 (2005).</li><li>Widdick, D. A., Eijlander, R. T., van Dijl, J. M., Kuipers, O. P., Palmer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Facile+Reporter+System+for+the+Experimental+Identification+of+Twin-Arginine+Translocation+(Tat)+Signal+Peptides+from+All+Kingdoms+of+Life.">A Facile Reporter System for the Experimental Identification of Twin-Arginine Translocation (Tat) Signal Peptides from All Kingdoms of Life.</a> Journal of Molecular Biology. 375 (3), 595-603 (2008).</li><li>Yuan, J., Cline, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastocyanin+and+the+33-kDa+subunit+of+the+oxygen-evolving+complex+are+transported+into+thylakoids+with+similar+requirements+as+predicted+from+pathway+specificity.">Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity.</a> Journal of Biological Chemistry. 269 (28), 18463-18467 (1994).</li><li>Kirchhoff, H., Borinski, M., Lenhert, S., Chi, L., B&#252;chel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transversal+and+lateral+exciton+energy+transfer+in+grana+thylakoids+of+spinach.">Transversal and lateral exciton energy transfer in grana thylakoids of spinach.</a> Biochemistry. 43 (45), 14508-14516 (2004).</li><li>Frielingsdorf, S., Jakob, M., Kl&#246;sgen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stromal+pool+of+TatA+promotes+Tat-dependent+protein+transport+across+the+thylakoid+membrane.">A stromal pool of TatA promotes Tat-dependent protein transport across the thylakoid membrane.</a> Journal of Biological Chemistry. 283 (49), 33838-33845 (2008).</li><li>Tu, C. -. J., Schuenemann, D., Hoffman, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+FtsY,+chloroplast+signal+recognition+particle,+and+GTP+are+required+to+reconstitute+the+soluble+phase+of+light-harvesting+chlorophyll+protein+transport+into+thylakoid+membranes.">Chloroplast FtsY, chloroplast signal recognition particle, and GTP are required to reconstitute the soluble phase of light-harvesting chlorophyll protein transport into thylakoid membranes.</a> Journal of Biological Chemistry. 274 (38), 27219-27224 (1999).</li></ol>

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 harvest...

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 border="0" cellpadding="0" cellspacing="0" style="width:1936px;" width="1936">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:324px;">Pisum sativum seeds</td>
			<td style="width:252px;">Seedway LLC, Hall, NY</td>
			<td style="width:292px;">8686 - Little Marvel</td>
			<td style="width:1068px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Miracloth</td>
			<td>Calbiochem, Gibbstown, NJ</td>
			<td>475855-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">80% Acetone</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>67-64-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blender with sharpened blades</td>
			<td>Waring Commercial</td>
			<td>BB155S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polytron 10-35</td>
			<td>Fischer Sci</td>
			<td>13-874-617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beckman J2-MC with JA 20 rotor</td>
			<td>Beckman-Coulter</td>
			<td>8043-30-1180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall RC-5B with HB-4 rotor</td>
			<td>Sorvall</td>
			<td>8327-30-1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K-Tricine</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>T0377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>50-70-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manganese Chloride</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>13446-34-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Sigma, Saint Louis, MO</td>
			<td>115-39-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 prot...

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 ...

This method can help answer key questions regarding the mechanisms and energetics of protein transport across chloroplast membranes, as well as the location of proteins within plastids. The main advantage of this technique for thylakoid protein targeting is that it allows for complete control of the thylakoid's environment during transport, including temperature, pH, ionic conditions, and precursor concentrations. Begin by soaking approximately 55 grams of peas in 400 milliliters of distilled water for three hours.Sow the soaked peas in a plastic tray in soil, covered with a thin layer of vermiculite, and grow for nine to 15 days as described in the text protocol. On the day of chloroplast isolation, prepare a continuous density gradient by centrifuging a mixture of 15 milliliters of Percoll and 15 milliliters of 2xGB buffer in a 15 milliliter polycarbonate high speed centrifuge tube at 30, 900 gs in a fixed angle rotor for 30 minutes at four degrees Celsius with the brake off. Then harvest approximately 25 grams of nine to 15 day old pea shoots.Add them to 95 milliliters of cold 1xGB buffer in a chilled blender, with sharpened blades. And homogenize. Filter this mixture through a minimum of two layers of cheese cloth, placed in a funnel over a chilled beaker.Centrifuge the filtrate using a 15 milliliter polycarbonate centrifuge tube in a swinging bucket rotor at 3, 000 gs for five minutes at four degrees Celsius. Discard the supernatant and use a paintbrush to gently resuspend the pellet in one milliliter of 1xGB. After that use a glass posture pipette to carefully layer the screen suspension on top of the Percoll gradient.Centrifuge it in a swinging bucket rotor at 8, 000 gs for 10 minutes at four degrees Celsius with the brake off. Using a posture pipette again carefully transfer the lower green band containing intact chloroplasts into a fresh 50 milliliter polycarbonate tube and add 30 milliliters of 1xIB buffer. Centrifuge the tube in a swinging bucket rotor at 1, 800 gs for five minutes at four degrees Celsius.After discarding the supernatant, gently resuspend the pellet in one milliliter of 1xIB with a paintbrush. Wash in 30 milliliters of 1xIB and centrifuge again under the same conditions. Resuspend the pellet after the final centrifugation in one milliliter of 1xIB.To determine chlorophyll concentration, mix 10 microliters of fully resuspended chloroplasts with five milliliters of 80%acetone. Filter through a 180 micrometer thick filter paper with 11 micrometer pore size and then determine the chlorophyll concentration by spectrophotometer. Dilute the chloroplast mixture to one milligram per milliliter using cold 1xIB.To start isolating the thylakoids when stromal extract is not necessary, such as cpTat transport, centrifuge intact chloroplasts in 1.5 milliliter tubes in a swinging bucket rotor at 1, 000 gs at four degrees Celsius for six minutes. After that, discard the supernatant and resuspend the pellet in hypotonic lysis buffer to achieve one milligram per milliliter with a micropipette. Incubate on ice in the dark with occasional mixing for 10 minutes.To recover thylokoids, centrifuge in a swinging bucket rotor at 3, 200 gs at four degrees Celsius for eight minutes. Wash the thylokoids twice by resuspending the pellet with one to two milliliters of lysis buffer and then centrifuging under the same conditions. After the final centrifugation, resuspend the pellet, in one milliliter of 1xIB buffer and determine the chlorophyll concentration in isolated thylokoids using a spectrophotometer as previously demonstrated.For pathways that require stromal extract recovery such as CPSec1 and CPSRP eliquate 600 microliters of isolated chloroplasts in a swinging bucket rotor and centrifuge at 1, 000 gs for six minutes at four degrees Celsius. For chloroplast lysis, we suspend the pellet with 600 microliters of hypotonic lysis buffer. Incubate on ice in the dark for 10 minutes with occasional mixing.Then centrifuge the tubes in a swinging bucket rotor at 3, 200 gs for eight minutes at four degrees Celsius. To save the stroma, transfer the light green to yellow supernatant to a new micro centrifuge tube and add an equal volume of 2x crude stromal buffer. To wash thylokoids, add two volumes of lysis buffer and centrifuge at 3, 200 gs for eight minutes at four degrees Celsius.Resuspend the pellet in 1xIB to achieve chlorophyll concentration of one milligram per milliliter and set aside on ice for later use in transport assays. Remove residual membranes from crude stroma by centrifuging the diluted crude stroma in a fixed angle rotor at 42, 000 gs for 30 minutes at four degrees Celsius. Then collect 95%of the volume from each tube making sure not to disturb the small yellow green pellet, while noting the volume taken from each tube.To prepare the concentrated stromal extract, pool the collected supernatants. Then use a four milliliter 30 kilodalton molecular weight cut-off concentrator to concentrate the extract five fold. To assay the cpTat transport in a 1.5 milliliter tube on ice mix the isolated thylokoids to a final chlorophyll concentration of 0.33 milligrams per milliliter, five millimolar ATP prepared in 1xIB, and eight millimolar DTT prepared in 1xIB.To initiate the transport, introduce substrate protein to the transport mix. Conduct the reaction by illuminating the suspension with 80 to 100 microeinsteins per square meter per second of photosynthetically active radiation for 10 minutes at room temperature. To stop the transport reaction, dilute the suspension eight fold with ice cold 1xIB.Centrifuge at 3, 200 gs in a micro centrifuge for five minutes at four degrees Celsius to recover thylokoids. After discarding the supernatant, resuspend the pellet in 120 microliters of ice cold 1xIB. To assay the CPSec1 or PSRP pathways mix the the following in a 1.5 milliliter tube on ice.Thylakoids to a final chlorophyll concentration of 0.33 milligrams per milliliter, 0.83 milligrams per milliliter chlorophyll equivalents of stromal extract, and five millimolar ATP prepared in 1xIB. Bring the reaction up to the desired volume with ice cold 1xIB and incubate on ice for 10 minutes. To initiate the transport introduce 10%volume by volume of substrate protein to this transport mix.Illuminate with 80 to 100 microeinsteins per square meter per second of photosynthetically active radiation for 10 minutes at room temperature. To stop the reaction, dilute the suspension eight fold with ice cold 1xIB. Centrifuge at 3, 200gs in a micro centrifuge for five minutes at four degrees Celsius.After discarding the supernatant, resuspend the pellet in 120 microliters of ice cold 1xIB. To remove residual substrate that has not been transported, digest external protein by adding six microliters of two milligrams per milliliter thermolysin and 10 millimolar calcium chloride in 1xIB. Incubate this protease reaction on ice for 40 minutes.Quench the protease by doubling the volume with 25 millimolar EDTA in 1xIB. Centrifuge at 3, 200 gs in a micro centrifuge for five minutes at four degrees Celsius to recover thylokoids. After removing the supernatant, resuspend recovered thylokoids in 120 microliters of five milimolar EDTA in 1xIB, and transfer to a new 1.5 milliliter tube.After centrifuging again at 3, 200 gs in a micro centrifuge for five minutes at four degrees Celsius, discard the supernatant and resuspend the pellet in an appropriate volume of laemmli sample buffer, supplemented with 10 millimolar EDTA. Place the samples in a boiling water bath for 10 minutes. Proceed to analyze the samples by SDS page.Transport of iOE17 protein through the cpTat pathway resulted in a size shift of two to three kilodaltons between the introduced substrate and the mature processed form. This is due to cleavage of the n terminal signal peptide indicating a successful cpTat transport. Thermolysin treatment leaves only the successfully transported and therefore proteus resistant mature band.Transport of OE33 precursor through the CPSec1 pathway, resulted in a size shift in the mature substrate, and proteus protection of the transported substrate. Insertion of LHCP precursor into the thylokoid membrane via the cpSRP pathway, evaluated through thermolysin digestion led to a size shift of 1.5 to two kilodaltons. This indicated successful membrane insertion, as the membrane protects the uncleaved of mature LHCP degradation product from complete proteolysis.After watching this video you should have a good understanding of how to isolate thylokoids and assay tranportivity through the energy dependent cpTat, cpSec 1 and cpSRP pathways. Once mastered this technique can be done in three to four hours when performed properly. Although not illustrated in this video we generally keep the overhead lights off in the lab when isolating and working with photosynthetically active chloroplasts and thylakoids.While attempting this procedure it's important to keep isolated chloroplast and thylakoids at four degrees Celsius. Remember to resuspend chloroplasts gently in the beginning. Avoid disturbing the Procoll gradient after the first centrifucation step.Additionally, percent input sample can be prepared in parallel with the transport reactions and run on the same gel to estimate the substrate transport. Don't forget, that working with radioactive substrates requires conscientious handling and proper PPE. After it's development, this technique paved the way for researchers in the field of protein targeting to explore the energetics, kinetics and unique mechanisms governing protein transport across thylakoid membranes.

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

Research

JoVE Journal

Biochemistry

10.1K Views.  University of California - Davis. This method can help answer key questions regarding the mechanisms and energetics of protein transport across chloroplast membranes, as well as the location of proteins within plastids. The main advantage of this technique for thylakoid protein targeting is that it allows for complete control of the thylakoid's environment during transport, including temperature, pH, ionic conditions, and precursor concentrations. Begin by soaking approximately 55 grams of peas in 400 milliliters of distilled...