title: Cytometric analysis of an epitope-tagged transit peptide bound to the chloroplast translocation apparatus.
abstract: Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope-tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His-S-SStp, contains a removable dual-epitope tag, His-S, at its N-terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser-scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C-terminal to the transit peptide, His-S-SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non-radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre- and post-import thermolysin treatment, photochemical cross-linking and organelle fractionation. The interaction was specific for the transit peptide 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 addition 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 translocation intermediate. In addition, direct visualization of His-S-SStp and Toc75 by confocal microscopy revealed a patch-like labeling, suggesting a co-ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast-binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.
authors.affiliation: The Graduate Group in Plant Physiology and Genetics, The University of Tennessee, Knoxville, TN 37996, USA.
authors.forename: C
authors.initials: C
authors.lastname: Subramanian
authors.forename: R
authors.initials: R
authors.lastname: Ivey
authors.forename: B D
authors.initials: BD
authors.lastname: Bruce
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D005434:Flow Cytometry; D006160:Guanosine Triphosphate / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013820:Thermolysin / Q000494:pharmacology
chemical_list: D004792:Enzyme Precursors; D000939:Epitopes; C430706:His-S-SStp protein, Pisum sativum; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D013820:Thermolysin