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Abstract
Translocation of outer membrane precursor proteins across the Escherichia coli inner membrane is severely hampered in lipid biosynthetic mutants with strongly reduced phosphatidylglycerol (PG) levels (De Vrije, T., De Swart, R. L., Dowhan, W., Tommassen, J., and De Kruijff, B. (1988) Nature 334, 173-175; Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271-280). Two independent methods were used to demonstrate that anionic lipids by virtue of their negative head-group charge are involved in membrane translocation of the precursor of the pore protein PhoE. Using a lipid transfer protein-based method we show that introduction from lipid vesicles of PG and other acidic phospholipids but not of phosphatidylcholine restores efficient translocation across the membrane of PG-depleted inner membrane vesicles. Moreover, translocation was found to be proportional to the PG content in vesicles isolated from strain HDL11 in which the PG content was altered by varying the synthesis of the PG-phosphate synthase.
Highlights
Introduction of Phospholipids into Inner MembraneVesicles-Inner membrane vesicles (50 nmol of phospholipid), isolated from strain, HDL11, grown in the absence of IPTG were incubated with small unilamellar vesicles (SUVs) (0-50 nmol of phospholipid) and - - pre PhoE r _-. . - . " " " --nonspecific lipid transfer protein (ns-LTP) in 200 p1 of 50 mM triethanolamineacetate,pH 7.5,250 mM sucrose, and 1 mg/ml bovine serum albumin (Sigma) for 25 min at 30°C
Translocation of in vitro-synthesized prePhoE is strongly reduced across inverted inner membrane vesicles isolated from an E. coli strain with low content of the negatively charged phospholipids PG and CL [23]
Inner membrane vesicles with variable levels of acidic phospholipids were isolated from strain HDL11, grown a t 10, 20, 30, or 50 p~ IPTG
Summary
Introduction of Phospholipids into Inner MembraneVesicles-Inner membrane vesicles (50 nmol of phospholipid), isolated from strain, HDL11, grown in the absence of IPTG were incubated with small unilamellar vesicles (SUVs) (0-50 nmol of phospholipid) and - - pre PhoE r _-. . - . " " " --ns-LTP (final concentration, 0.12 mg/ml) in 200 p1 of 50 mM triethanolamineacetate,pH 7.5,250 mM sucrose, and 1 mg/ml bovine serum albumin (Sigma) for 25 min at 30°C. Using a lipid transfer protein-based method we show that introduction from lipid vesicles of PG and other acidic phospholipids but not of phosphatidylcholine restores efficient translocation across the membrane of PG-depleted inner membrane vesicles.
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