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J. Biol. Chem., Vol. 280, Issue 50, 41165-41170, December 16, 2005

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The Thylakoid pH/ Are Not Required for the Initial Stages of Tat-dependent Protein Transport in Tobacco Protoplasts^*

From the Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

The twin-arginine translocation (Tat) system transports folded proteins across the chloroplast thylakoid membrane and bacterial plasma membrane. In vitro import assays have pointed to a key role for the thylakoid pH in the initial assembly of the full translocon from two subcomplexes; more generally, the pH is believed to provide the overall driving force for translocation. Here, we have studied the role of the pH in vivo by analyzing the translocation of Tat substrates in transfected tobacco protoplasts. We show that the complete maturation of the precursor of the 23-kDa lumenal protein (pre-23K) and of a fusion of the 23K presequence linked to green fluorescent protein (pre-GFP) are unaffected by dissipation of the pH. High level expression of Tat substrates in protoplasts has recently been shown to result in "translocation reversal" in that a large proportion of a given substrate is partially translocated across the thylakoid membrane, processed to the mature size, and returned to the stroma. However, the efficiency of translocation of pre-23K is undiminished in the absence of the pH and/or , and the rate and extent of maturation of both pre-23K and pre-GFP by the lumen-facing processing peptidase is similarly unaffected. These data demonstrate that the proton motive force is not required for the functional assembly of the Tat translocon and the initial stages of translocation in higher plant chloroplasts in vivo. We conclude that unknown factors play an influential role in both the mechanism and energetics of this system under in vivo conditions.

Received for publication, August 22, 2005

^* This work was supported by Biotechnology and Biological Sciences Research Council Grants C15308 [GenBank] and C15310 [GenBank] (to C. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 44-2476-523557; Fax: 44-2476-523568; E-mail: Crobinson{at}bio.warwick.ac.uk.

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