Abstract
Chloroplasts contain about 3000 proteins, with approximately 400 of them located in the chloroplast envelope membranes, 1300 in the soluble stroma, and 1300 in the thylakoid membranes. Most of them are encoded by nuclear genes and translated as precursor proteins in the cytosol before their transport into chloroplasts. As a tool to control and characterize their import into plastids, we here describe an assay for in vitro protein import with isolated pea chloroplasts.
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References
Bölter B, Soll J (2016) Once upon a time—chloroplast protein import research from infancy to future challenges. Mol Plant 9:798–812
Miras S, Salvi D, Piette L et al (2007) Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts. J Biol Chem 282(40):29482–29492
Rossig C, Reinbothe C, Gray J et al (2013) Three proteins mediate import of transit sequence-less precursors into the inner envelope of chloroplasts in Arabidopsis thaliana. Proc Natl Acad Sci U S A 110(49):19962–19967
Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8(5):978–984
Bruley C, Dupierris V, Salvi D et al (2012) AT_CHLORO: a chloroplast protein database dedicated to sub-Plastidial localization. Front Plant Sci 3:205
Waegemann K, Soll J (1995) Characterization and isolation of the chloroplast protein import machinery. Methods Cell Biol 50:255–267
Block MA, Tewari AK, Albrieux C et al (2002) The plant S-adenosyl-l-methionine:mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes. Eur J Biochem 269:240–248
Joyard J, Billecocq A, Bartlett SG et al (1983) Localization of polypeptides to the cytosolic side of the outer envelope membrane of spinach chloroplasts. J Biol Chem 258:10000–10006
Smeekens S, Bauerle C, Hageman J et al (1986) The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell 46:365–375
Hardré H, Kuhn L, Albrieux C et al (2014) The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes. Front Plant Sci 5:203. https://doi.org/10.3389/fpls.2014.00203
Li HM, Moore T, Keegstra K (1991) Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts. Plant Cell 7:709–717
Acknowledgments
This work was supported by the French National Research Agency (ANR-13-ADAP-0008 Reglisse and ANR-10-LABEX-04 GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology).
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Block, M.A. (2018). In Vitro Protein Import into Isolated Chloroplasts. In: Maréchal, E. (eds) Plastids. Methods in Molecular Biology, vol 1829. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8654-5_11
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DOI: https://doi.org/10.1007/978-1-4939-8654-5_11
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-8654-5
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