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.