The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.
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.
Inoue
K
K
MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.
Demel
R
R
de Kruijff
B
B
Keegstra
K
K
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
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