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 bipartit e transit peptide. The N-terminal portion of the Toc75 transit peptide is s ufficient to target the protein to the stromal space of chloroplasts. We pr epared a 45 amino-acid peptide containing the stromal targeting domain of t he Toc75 transit peptide in Escherichia coli, using the intein-mediated sys tem, and purified it by reverse-phase HPLC. Its identity was confirmed by N -terminal amino-acid sequencing and matrix assisted laser desorption ioniza tion mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosp hatidylglycerol 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 t o intact chloroplasts as effectively as did a bacterially produced precurso r or the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The pepti de also inhibited import of radiolabeled precursors into isolated chloropla sts, however, to a lesser extent than did nonlabeled precursor or the small subunit of 1,5-bisphosphate carboxylase/oxygenase.