The thylakoid delta pH/delta psi are not required for the initial stages of Tat-dependent protein transport in tobacco protoplasts.
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 delta pH in the initial assembly of the full translocon from two subcomplexes; more generally, the delta pH is believed to provide the overall driving force for translocation. Here, we have studied the role of the delta 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 delta 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 delta pH and/or delta psi, 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.
Di Cola
Alessandra
A
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Bailey
Shaun
S
Robinson
Colin
C
D001419:Bacteria / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D015696:Gene Products, tat / Q000737:chemistry*; D017343:Genes, Plant; D015756:Genes, tat; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000378:metabolism; D017510:Protein Folding; D021381:Protein Transport; D018892:Proton-Motive Force; D011523:Protoplasts; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020524:Thylakoids / Q000378:metabolism*; D013997:Time Factors; D014026:Nicotiana / Q000378:metabolism*; D014162:Transfection
D015696:Gene Products, tat; D010455:Peptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins