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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
Pisum sativum seedsSeedway LLC, Hall, NY8686 - Little Marvel
MiraclothCalbiochem, Gibbstown, NJ475855-1
80% AcetoneSigma, Saint Louis, MO67-64-1
Blender with sharpened bladesWaring CommercialBB155S
Polytron 10-35Fischer Sci13-874-617
PercollSigma, Saint Louis, MOGE17-0891-01
Beckman J2-MC with JA 20 rotorBeckman-Coulter8043-30-1180
Sorvall RC-5B with HB-4 rotorSorvall8327-30-1016
100 mM dithiothreitol (DTT) in 1xIBSigma, Saint Louis, MO12/3/83Can be frozen in aliquots for future use
200 mM MgATP in 1xIBSigma, Saint Louis, MO74804-12-9Can be frozen in aliquots for future use
Thermolysin in 1xIB (2mg/mL)Sigma, Saint Louis, MO9073-78-3Can be frozen in aliquots for future use
HEPESSigma, Saint Louis, MOH3375
K-TricineSigma, Saint Louis, MOT0377
SorbitolSigma, Saint Louis, MO50-70-4
Magnesium ChlorideSigma, Saint Louis, MO7791-18-6
Manganese ChlorideSigma, Saint Louis, MO13446-34-9
EDTASigma, Saint Louis, MO60-00-4
BSASigma, Saint Louis, MO9048-46-8
TrisSigma, Saint Louis, MO77-86-1
SDSSigma, Saint Louis, MO151-21-3
GlycerolSigma, Saint Louis, MO56-81-5
Bromophenol BlueSigma, Saint Louis, MO115-39-9
B-MercaptoethanolSigma, Saint Louis, MO60-24-2

References

  1. Ellis, R. Chloroplast protein synthesis: principles and problems. Sub-cellular biochemistry. 9, 237 (1983).
  2. Li, H. -. m., Chiu, C. -. C. Protein transport into chloroplasts. Annual review of plant biology. 61, (2010....

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