SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.
In order to analyze the information content of a chloroplast transit sequence, we have constructed and analyzed by in vitro assays seven substitution and 20 deletion mutants of the ferredoxin transit sequence. The N-terminal part and the C-terminal part are important for targeting, and in addition the C-terminal region is required for processing. A third region is important for translocation but not for the initial interaction with the envelope. A fourth region is less essential for in vitro import. Purified precursors were tested for their ability to compete for the in vitro import of radiolabeled wild-type precursor, which confirmed the important role in chloroplast recognition of both the N- and the C-terminal domain of the transit sequence. Monolayer experiments showed that the N terminus was mainly involved in the insertion into mono-galactolipid-containing lipid surfaces whereas the C terminus mediates the recognition of negatively charged lipids. A sequence comparison to other transit sequences suggests that the domain structure of the ferredoxin transit sequence can be extended to these sequences and thus reveals a general structural design of transit sequences.
The aggregation of octadecyl rhodamine 101 (Rh101C 18 ) and a Rh101-labeled transmembrane peptide (Rh101WALP16) solubilized in lipid phases was studied. Different lipid phases in excess of water were formed with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or 1,2-dierucoyl-sn-glycero-3-phosphochline (DErPC). Rh101 was covalently attached with its carboxyl group to the C-terminal ethanolamine. Time-resolved polarized fluorescence spectroscopy was used to determine the fluorescence relaxation and anisotropy at molar ratios ranging from 1 Rh101derivative per 5000 lipids (1:5000) to 1 Rh101-derivative per 50 lipids (1:50). At concentrations of the Rh101derivatives exceeding about 1 mol %, the fluorescence intensity is quenched, and the fluorescence lifetime is significantly decreased. This is compatible with electronic energy transfer to ground-state dimers of Rh101groups. The time-resolved fluorescence decays were analyzed by analytical models of donor-acceptor electronic energy transfer. The models account for energy transfer between Rh101 monomers (donors) and Rh101dimers (acceptors), spatially distributed in one and two dimensions. For the amphiphilic Rh101C 18 molecules solubilized in DMPC, DOPC, and DErPC, the fluorescence relaxation is very well described as energy transfer in lipid vesicles between donors and acceptors, distributed on the inner and outer surfaces of the bilayer. For statistical reasons, pairs of molecules in contact are more likely to appear at high concentrations. Hereafter these are referred to as statistical dimers. It is found that the dimer concentration of Rh101C 18 in the bilayer of the various lipids, is slightly above that calculated for statistical dimers. In contrast, the dimerization of Rh101WALP16 is five-to 10-fold increased, as compared to the expected concentration of statistical dimers. This suggests that the transmembrane peptide has an inherent affinity for aggregation in lipid bilayers formed by DMPC, DOPC, and DErPC. The fluorescence relaxation of Rh101WALP16 in DMPC and DOPC is very well described as donor-acceptor energy transfer across the lipid bilayer of vesicles. However, the fluorescence relaxation of Rh101WALP16 in DErPC is not compatible with energy transfer in lipid bilayers. Instead, a better description is achieved by assuming that the Rh101WALP16 molecules are aggregated along parallel lines. Such a spatial distribution is compatible with recent studies showing a reversed hexagonal phase (H II ) that appears at high WALP16 concentrations in DErPC (J. A. Killian et al., Biochemistry 1996, 35, 1037.
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