Cytometric analysis of an epitope-tagged transit peptide bound to the chloroplast translocation apparatus

Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. H owever, the mechanism by which transit peptides engage the translocation ap paratus has not been investigated. To analyse this interaction, we have dev eloped a novel epitope-tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His-S-S Stp, contains a removable dual-epitope tag, His-S, at its N-terminus that p ermits both rapid purification via immobilized metal affinity chromatograph y and detection by blotting, flow cytometry and laser-scanning confocal mic roscopy. Unlike other chimeric precursors, which place the passenger protei n C-terminal to the transit peptide, His-S-SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This ear ly translocation intermediate allowed non-radioactive detection using fluor escent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pr e- and post-import thermolysin treatment, photochemical cross-linking and o rganelle fractionation. The interaction was specific for the transit peptid e since His-S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry , showing saturation of binding yet only slight ATP-dependence. The additio n of GTP showed inhibition of the binding of His-S-SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocat ion intermediate. In addition, direct visualization of His-S-SStp and Toc75 by confocal microscopy revealed a patch-like labeling, suggesting a co-ord inate localization to discrete regions on the chloroplast envelope. These f indings represent the first direct visualization of a transit peptide inter acting with the chloroplast translocation apparatus. Furthermore, identific ation of a chloroplast-binding intermediate may provide a novel tool to dis sect interactions between a transit peptide and the chloroplast translocati on apparatus.