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Protocol

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Materials

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Biochemistry

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

Published: September 28th, 2018

DOI:

10.3791/58393

1Department of Plant Biology, University of California - Davis

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....

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1. Initial Materials

  1. Soak approximately 55 g of peas for 3 hours in 400 mL of distilled water, and then sow in a plastic tray (35 cm x 20 cm x 6 cm) in soil covered with thin layer of vermiculite.
  2. Grow the tray of peas at 20 °C under 12/12 h light/dark (50 µE/m2s) cycle for 9 to 15 days.
  3. Prepare protein substrate according to a preferred method.
    Note: We have prepared protein substrates using a variety of methods, including 1) in vitro transcription fro.......

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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.......

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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.......

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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

....

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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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