IN-VITRO INTERACTION BETWEEN A CHLOROPLAST TRANSIT PEPTIDE AND CHLOROPLAST OUTER ENVELOPE LIPIDS IS SEQUENCE-SPECIFIC AND LIPID CLASS-DEPENDENT

Interaction of artificial lipid bilayers (liposomes) with the purified transit peptide (SS-tp) of the precursor form of the small subunit fo r ribulose-2,5-bisphosphate carboxylase/oxygenase (prSSU) has been stu died using a vesicle-disruption assay (calcein dye release) and electr on microscopy. Employing purified forms of Escherichia coli-expressed prSSU, mature small subunit, glutathione S-transferase-transit peptide fusion protein, and SS-tp in dye release studies demonstrated that li pid interaction is mediated primarily through the transit peptide. Usi ng chemically synthesized peptides (20-mers), the lipid-interacting do main of the transit peptide was partially mapped to the C-terminal 20 amino acids of the transit peptide. Peptides corresponding to other re gions of the transit peptide and control peptides promoted significant ly less calcein release. Interaction between the transit peptide and t he bilayer was very rapid and could not be resolved by stopped-flow fl uorometry with a mixing time of <50 ms. Interaction between the peptid es and bilayer was also lipid class-dependent. Disruption occurred onl y when the bilayer contained the galactolipid monogalactosyldiacylglyc erol (MGDG). The extent of bilayer disruption directly correlated with the relative concentration of MGDG in the liposome, with maximum calc ein release occurring in 20 mol % MGDG liposomes. Lipid bilayers with greater than 20 mol % MGDG could not be achieved as determined by calc ein entrapment. Electron microscopy of the liposomes before and after addition of the transit peptide suggested that the transit peptide ind uced a dramatic reorganization of lipids, These results are discussed in light of a possible mechanism for the early steps in protein transp ort that may involve polymorphic changes in the envelope membrane orga nization to include localized non-bilayer H-II structures.