title: The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.
abstract: Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.
authors.affiliation: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: K
authors.initials: K
authors.lastname: Inoue
authors.forename: R
authors.initials: R
authors.lastname: Demel
authors.forename: B
authors.initials: B
authors.lastname: de Kruijff
authors.forename: K
authors.initials: K
authors.lastname: Keegstra
mesh_terms: D002736:Chloroplasts / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D018532:Pisum sativum; D010446:Peptide Fragments / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; C103933:OEP75 protein precursor, plant; D010446:Peptide Fragments; D010940:Plant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012273:Ribulose-Bisphosphate Carboxylase