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Targeting of EGFP chimeras within chloroplasts

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Abstract

We have tested the potential of EGFP, a derivative of the green fluorescent protein (GFP), as a passenger protein for the analysis of protein transport processes across the thylakoid membranes in chloroplasts. In contrast to the majority of fusion proteins commonly used in such studies, EGFP is not of plant origin and can therefore be assumed to behave like a "neutral" passenger protein that is unaffected by any internal plant regulatory circuits. Our in vitro transport experiments clearly demonstrate that EGFP is a suitable passenger protein that can be correctly targeted either to the stroma or to the thylakoid lumen if fused to the appropriate transit peptide. The transport of EGFP across the thylakoid membrane shows, however, a clear pathway preference. While the protein is efficiently targeted by the ΔpH/TAT pathway, transport by the Sec pathway is barely detectable, either with isolated thylakoids or with intact chloroplasts. This pathway specificity suggests that EGFP is folded immediately after import into the chloroplast stroma, thus preventing further translocation across the thylakoid membrane by the Sec translocase. The data obtained provide a good basis for the development of molecular tools for transport studies using EGFP as a passenger protein. Furthermore, plant lines expressing corresponding EGFP chimeras are expected to allow in vivo studies on the transport and sorting mechanisms involved in the biogenesis of the chloroplast.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (grants SFB 184, SFB 363 and KL862/1-1)

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Authors and Affiliations

  1. Institut für Pflanzenphysiologie, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 10, 06120, Halle (Saale), Germany

    J. P. Marques, I. Dudeck & R. B. Klösgen

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  1. J. P. Marques
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  2. I. Dudeck
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  3. R. B. Klösgen
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Correspondence to R. B. Klösgen.

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Communicated by R. Hagemann

The first two authors contributed equally to this work

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Marques, J.P., Dudeck, I. & Klösgen, R.B. Targeting of EGFP chimeras within chloroplasts. Mol Gen Genomics 269, 381–387 (2003). https://doi.org/10.1007/s00438-003-0846-y

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