Nuclear‐encoded Protein Import into Chloroplasts: Methods


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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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. [doi: 10.1002/9780470015902.a0002620.pub2]