Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays
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. We report these methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.
- Research Organization:
- Univ. of California, Davis, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB)
- DOE Contract Number:
- SC0017035
- OSTI ID:
- 1632709
- Journal Information:
- Journal of Visualized Experiments, Vol. 2018, Issue 139; ISSN 1940-087X
- Publisher:
- MyJoVE Corp.
- Country of Publication:
- United States
- Language:
- English
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