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Vol. 20, Issue 7, 2060-2069, April 1, 2009

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Clustering of C-Terminal Stromal Domains of Tha4 Homo-oligomers during Translocation by the Tat Protein Transport System

Plant Molecular and Cellular Biology Program and Department of Horticultural Sciences, University of Florida, Gainesville, FL 32611

Submitted December 10, 2008; Revised January 27, 2009; Accepted January 28, 2009
Monitoring Editor: Reid Gilmore

The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor–receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.

* Present address: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056.

Address correspondence to: Kenneth Cline (kcline{at}ufl.edu)

Abbreviations used: APH, amphipathic helix; BMOE, bis-maleimidoethane; BM(PEO)₃, 1,11-bis-maleimido-triethyleneglycol; C-tail, carboxy-terminal loosely structured domain of Tha4; CuP, Cu⁺⁺ 1,10-o-phenanthroline; DSP, dithiobis(succinimidyl propionate); DTT, dithiothreitol; IB, import buffer; NEM, N-ethyl maleimide; Tat, twin arginine translocation; TMD, transmembrane domain, tpOE17, 31-amino acid synthetic peptide of the lumen-targeting sequence of the 17-kDa subunit of the oxygen evolving complex of photosystem II.

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