Chapter title |
Preparation of Membrane Fractions (Envelope, Thylakoids, Grana, and Stroma Lamellae) from Arabidopsis Chloroplasts for Quantitative Proteomic Investigations and Other Studies
|
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Chapter number | 8 |
Book title |
Plant Membrane Proteomics
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7411-5_8 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7409-2, 978-1-4939-7411-5
|
Authors |
Lucas Moyet, Daniel Salvi, Martino Tomizioli, Daphné Seigneurin-Berny, Norbert Rolland |
Abstract |
Chloroplasts are semiautonomous organelles found in plants and protists. They are surrounded by a double membrane system, or envelope. These envelope membranes contain machineries to import nuclear-encoded proteins, and transporters for ions or metabolites, but are also essential for a range of plastid-specific metabolisms. The inner membrane surrounds a stroma, which is the site of the carbon chemistry of photosynthesis. Chloroplasts also contain an internal membrane system, or thylakoids, where the light phase of photosynthesis occurs. The thylakoid membranes themselves have a bipartite structure, consisting of grana stacks interconnected by stroma lamellae. These thylakoid membranes however form a continuous network that encloses a single lumenal space. Chloroplast-encoded or targeted proteins are thus addressed to various sub-compartments that turn out to be flexible systems and whose main functions can be modulated by alterations in the relative levels of their components. This article describes procedures developed to recover highly purified chloroplast membrane fractions (i.e., envelope, crude thylakoid membranes, as well as the two main thylakoid subdomains, grana and stroma lamellae), starting from Percoll-purified Arabidopsis chloroplasts. Immunological markers are also listed that can be used to assess the purity of these fractions and reveal specific contaminations by other plastid membrane compartments. The methods described here are compatible with chloroplast proteome dynamic studies relying on targeted quantitative proteomic investigations. |
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