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JXB Advance Access published online on June 18, 2004
Journal of Experimental Botany, doi:10.1093/jxb/erh191
© 2004 by Oxford University Press
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Received February 25, 2004; accepted May 4, 2004
© 2004 Society for Experimental Biology

RESEARCH PAPER

In vivo transport of folded EGFP by the {Delta}pH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana

João Pedro Marques 1*, Martin Hartmut Schattat 1, Gerd Hause 2, Ingrid Dudeck 1, and Ralf Bernd Klösgen 1

1 Institut für Pflanzenphysiologie, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 10, D-06120 Halle (Saale), Germany
2 Biozentrum der Universität, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, D-06120 Halle (Saale), Germany

* To whom correspondence should be addressed. E-mail: marques{at}pflanzenphys.uni-halle.de.


  Abstract

Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the {Delta}pH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the {Delta}pH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the {Delta}pH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the {Delta}pH/TAT pathway.

Keywords: Chloroplast, green fluorescent protein (GFP), {Delta}pH/TAT pathway, protein transport, thylakoid membrane


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