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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 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.
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-yie....
1. Initial Materials
To gauge amount of substrate successfully transported, it is useful to include one or more "percent input" lanes. For the data presented below, 10% of the final transport reaction without thylakoids was used. This "percent input" 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.......
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.......
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
....Name | Company | Catalog Number | Comments |
Pisum sativum seeds | Seedway LLC, Hall, NY | 8686 - Little Marvel | |
Miracloth | Calbiochem, Gibbstown, NJ | 475855-1 | |
80% Acetone | Sigma, Saint Louis, MO | 67-64-1 | |
Blender with sharpened blades | Waring Commercial | BB155S | |
Polytron 10-35 | Fischer Sci | 13-874-617 | |
Percoll | Sigma, Saint Louis, MO | GE17-0891-01 | |
Beckman J2-MC with JA 20 rotor | Beckman-Coulter | 8043-30-1180 | |
Sorvall RC-5B with HB-4 rotor | Sorvall | 8327-30-1016 | |
100 mM dithiothreitol (DTT) in 1xIB | Sigma, Saint Louis, MO | 12/3/83 | Can be frozen in aliquots for future use |
200 mM MgATP in 1xIB | Sigma, Saint Louis, MO | 74804-12-9 | Can be frozen in aliquots for future use |
Thermolysin in 1xIB (2mg/mL) | Sigma, Saint Louis, MO | 9073-78-3 | Can be frozen in aliquots for future use |
HEPES | Sigma, Saint Louis, MO | H3375 | |
K-Tricine | Sigma, Saint Louis, MO | T0377 | |
Sorbitol | Sigma, Saint Louis, MO | 50-70-4 | |
Magnesium Chloride | Sigma, Saint Louis, MO | 7791-18-6 | |
Manganese Chloride | Sigma, Saint Louis, MO | 13446-34-9 | |
EDTA | Sigma, Saint Louis, MO | 60-00-4 | |
BSA | Sigma, Saint Louis, MO | 9048-46-8 | |
Tris | Sigma, Saint Louis, MO | 77-86-1 | |
SDS | Sigma, Saint Louis, MO | 151-21-3 | |
Glycerol | Sigma, Saint Louis, MO | 56-81-5 | |
Bromophenol Blue | Sigma, Saint Louis, MO | 115-39-9 | |
B-Mercaptoethanol | Sigma, Saint Louis, MO | 60-24-2 |
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