INFLUENCE OF THE SIGNAL SEQUENCE AND CHAPERONE SECB ON THE INTERACTION BETWEEN PRECURSOR PROTEIN PREPHOE AND PHOSPHOLIPIDS

To investigate in a direct way the interaction between a precursor pro tein and phospholipids, monolayer studies were performed using the pur ified precursor of Escherichia coli outer-membrane protein PhoE. It wa s demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole(2)GroPGro) and dioleoylglycerophos phoethanolamine (Ole(2)GroPEtn), this insertion was mainly driven by h ydrophobic forces. Compared with previous results obtained with PhoE s ignal peptide, the full-length precursor protein does not show the spe cific interaction with acidic lipids. PrePhoE inserted into a Ole(2)Gr oPGro monolayer occupies an area of 28 +/- 30 nm(2)/molecule, which is approximately 10-fold larger than the area occupied by the PhoE signa l peptide. The purified mature PhoE protein has a lower capacity to in sert into Ole(2)GroPGro and Ole(2)GroPEtn monolayers and is, in contra st to prePhoE, fully accessible to proteinase K after interacting with a Ole(2)GroPGro monolayer. The results demonstrate that in the contex t of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers. It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB . Complexation with SecB increases the insertion of (pre)PhoE into aci dic lipid monolayers. The high lipid affinity of prePhoE was also demo nstrated by vesicle-binding experiments which showed that SecB dissoci ates from the SecB-prePhoE complex upon binding of the precursor to th e bilayer. The implications of these findings for preprotein transloca tion are discussed and in addition some extrapolations to the insertio n of PhoE into the outer membrane are made.