Nuclear‐encoded Protein Import into Chloroplasts: Methods

Abstract

Chloroplasts carry out essential processes, for example photosynthesis. They are derived from an endosymbiotic event, in which an ancient cyanobacterium was engulfed by an eukaryotic ancestor. During evolution, most of the genes have been transferred to the nucleus. Consequently, >98% of all plastid proteins are translated on cytosolic ribosomes and posttranslationally targeted to and imported into the organelle. Targeting is assisted by cytosolic proteins, which keep them in an import‐competent state. At the chloroplast, many proteins have to conquer the barrier of the chloroplast envelopes. This process is mediated by two complexes in the outer (Toc) and inner (Tic) envelope. Most proteins destined for inner compartments contain a cleavable N‐terminal transit peptide, whereas most outer envelope components insert into the membrane without such a targeting peptide. Investigation of the import of nuclear‐encoded proteins into chloroplasts is an essential field of plant science. To throw light on this very basic process many different methods have been established.

Key Concepts:

  • Studying the in vitro import behaviour of chloroplastic preproteins gives essential insight into this fundamental process.

  • Analysis of mutants in model organisms provides information about the in vivo function of proteins proposed to be involved into translocation.

Keywords: chloroplasts; precursor proteins; import; chemical crosslinking

Figure 1.

Sequence of experimental steps to study protein import into chloroplasts.

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References

Agne B and Kessler F (2009) Protein transport in organelles: the Toc complex way of preprotein import. FEBS Journal 276: 1156–1165.

Benz JP, Soll J and Bölter B (2009) Protein transport in organelles: the composition, function and regulation of the Tic complex in chloroplast protein import. FEBS Journal 276: 1166–1176.

Bölter B, May T and Soll J (1998) A protein import receptor in pea chloroplasts, Toc86, is only a proteolytic fragment of a larger polypeptide. FEBS Letters 441: 59–62.

Firlej‐Kwoka E, Strittmatter P, Soll J and Bölter B (2008) Import of preproteins into the chloroplast inner envelope membrane. Plant Molecular Biology 68: 505–509.

Hofmann N and Theg S (2003) Physcomitrella patens as a model for the study of chloroplast protein transport: conserved machineries between vascular and non‐vascular plants. Plant Molecular Biology 53: 321–632.

Inoue H and Akita M (2008) Three sets of translocation intermediates are formed during the early stage of protein import into chloroplasts. Journal of Biological Chemistry 283: 7491–7502.

Jarvis P (2008) Targeting of nucleus‐encoded proteins to chloroplasts in plants. New Phytology 179: 257–285.

Jarvis P, Li LJ, Li H et al. (1998) An Arabidopsis mutant defective in the plastid general protein import apparatus. Science 282: 100–103.

Küchler M, Decker S, Hörmann F, Soll J and Heins L (2002) Protein import into chloroplasts involves redox‐regulated proteins. EMBO Journal 21: 6136–6145.

Lalonde S, Ehrhardt DW, Loqué D et al. (2008) Molecular and cellular approaches for the detection of protein‐protein interactions: latest techniques and current limitations. Plant Journal 53: 610–635.

Lang D, Zimmer AD, Rensing SA and Reski R (2008) Exploring plant biodiversity: the Physcomitrella genome and beyond. Trends in Plant Science 13: 524–549.

Lübeck J, Heins L and Soll J (1996) A nuclear‐coded chloroplastic inner envelope membrane protein uses a soluble sorting intermediate upon import into the organelle. Journal of Cell Biology 137: 1279–1286.

Perry SE and Keegstra KC (1994) Envelope membrane proteins that interact with chloroplastic precursor proteins. Plant Cell 6: 93–105.

Reisinger V and Eichacker LA (2007) How to analyze protein complexes by 2D blue native SDS‐PAGE. Proteomics 7(suppl. 1): 6–16.

Schleiff E, Soll J, Küchler M, Kühlbrandt W and Harrer R (2003) Characterization of the translocon of the outer envelope of chloroplasts. Journal of Cell Biology 160: 541–551.

Schleiff E, Soll J, Sveshnikova N et al. (2002) Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34. Biochemistry 41: 1934–1946.

Stengel A, Soll J and Bölter B (2008) Protein import into chloroplasts: new aspects of a well‐known topic. Biological Chemistry 388: 765–772.

Waegemann K and Soll J (1995) Characterization and isolation of the chloroplast protein import machinery. Methods in Cell Biology 50: 255–267.

Further Reading

Bölter B and Soll J (2007) Import of plastid precursor proteins into chloroplasts. In: Van der Giezen M (ed.) Methods in Molecular Biology: Protein Targeting Protocols, pp. 195–206. Totowa: Humana Press.

Cline K and Henry R (1996) Import and routing of nucleus‐encoded chloroplast proteins. Annual Review of Cell and Developmental Biology 12: 1–26.

Heins L and Soll J (1998) Mixing the prokaryotic and the eukaryotic. Current Biology 8: 215–217.

Kubis SE, Lilley KS and Jarvis P (2008) Isolation and preparation of chloroplasts from Arabidopsis thaliana plants. In: Posch A (ed.) Methods in Molecular Biology, 2D PAGE: Sample Preparation and Fractionation, pp. 171–186. Totowa: Humana Press.

Soll J (ed.) (1998) Protein Trafficking in Plant cells. Dordrecht: Kluwer Academic.

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Soll, Jürgen, and Bölter, Bettina(Apr 2010) Nuclear‐encoded Protein Import into Chloroplasts: Methods. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002620.pub2]