The interaction between SStp, the transit peptide of the precursor protein to the small subunit of Rubisco (prSSU) and two Hsp70 molecular chaperones, Escherichia coli DnaK and pea (Pisum sativum) CSS1, was investigated in detail. Two statistical analyses were developed and used to investigate and predict regions of SStp recognized by DnaK. Both algorithms suggested that DnaK would have high affinity for the N terminus of SStp, moderate affinity for the central region, and low affinity for the C terminus. Furthermore, both algorithms predicted this affinity pattern for >75% of the transit peptides analyzed in the chloroplast transit peptide (CHLPEP) database. In vitro association between SStp and these Hsp70s was confirmed by three independent assays: limited trypsin resistance, ATPase stimulation, and native gel shift. Finally, synthetic peptides scanning the length of SStp and C-terminal deletion mutants of SStp were used to experimentally map the region of greatest DnaK affinity to the N terminus. CSS1 displayed a similar affinity for the N terminus of SStp. The major stromal Hsp70s affinity for the N terminus of SStp and other transit peptides supports a molecular motor model in which the chaperone functions as an ATP-dependent translocase, committing chloroplast precursor proteins to unidirectional movement across the envelope.

Interaction of artificial lipid bilayers (liposomes) with the purified transit peptide (SS-tp) of the precursor form of the small subunit for ribulose-2,5-bisphosphate carboxylase/oxygenase (prSSU) has been studied using a vesicle-disruption assay (calcein dye release) and electron 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 lipid interaction is mediated primarily through the transit peptide. Using chemically synthesized peptides (20-mers), the lipid-interacting domain of the transit peptide was partially mapped to the C-terminal 20 amino acids of the transit peptide. Peptides corresponding to other regions of the transit peptide and control peptides promoted significantly less calcein release. Interaction between the transit peptide and the bilayer was very rapid and could not be resolved by stopped-flow fluorometry with a mixing time of <50 ms. Interaction between the peptides and bilayer was also lipid class-dependent. Disruption occurred only when the bilayer contained the galactolipid monogalactosyldiacylglycerol (MGDG). The extent of bilayer disruption directly correlated with the relative concentration of MGDG in the liposome, with maximum calcein release occurring in 20 mol % MGDG liposomes. Lipid bilayers with greater than 20 mol % MGDG could not be achieved as determined by calcein entrapment. Electron microscopy of the liposomes before and after addition of the transit peptide suggested that the transit peptide induced a dramatic reorganization of lipids. These results are discussed in light of a possible mechanism for the early steps in protein transport that may involve polymorphic changes in the envelope membrane organization to include localized non-bilayer H_II structures.