Format

Send to

Choose Destination
J Vis Exp. 2018 Sep 28;(139). doi: 10.3791/58393.

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

Author information

1
Department of Plant Biology, University of California - Davis.
2
Department of Plant Biology, University of California - Davis; smtheg@ucdavis.edu.

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.

PMID:
30320750
PMCID:
PMC6235370
[Available on 2020-09-28]
DOI:
10.3791/58393
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for MyJove Corporation
Loading ...
Support Center