Transit sequence-dependent binding of the chloroplast precursor protein ferredoxin to lipid vesicles and its implications for membrane stability

AbstractThe binding of the transit peptide (trfd) and precursor of the chloroplast protein ferredoxin (prefd) to large unilamellar lipid vesicles was investigated in relation to the lipid composition of the bilayer. Prefd binds with a dissociation constant of 0.27 μM to vesicles with a composition corresponding to the chloroplast envelope outer membrane. Binding is mediated by the transit sequence. From an analysis of binding to vesicles containing the individual lipid components it could be concluded that anionic lipids are mainly responsible for binding, emphasizing the importance of electrostatics for the transit sequence-lipid interaction. Binding is also mediated by the specific chloroplast glycolipid monogalactosyldiacylglycerol. Monolayer experiments revealed that in this case a more extended domain of the transit sequence inserts into the lipid layer. Precursor binding does not result in a loss of vesicle barrier function. However, high concentrations of trfd do cause release of vesicle-enclosed carboxyfluorescein. The results are discussed in the light of the chloroplast protein import process, with special emphasis on the role of monogalactosyldiacylglycerol

The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids

AbstractImport of proteins into chloroplasts depends on an N-terminal transit sequence. Transit sequences contain little primary sequence similarity and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of the transit sequence, we studied the transit peptide of preferredoxin (trfd) by circular dichroism. In buffer, trfd is in a random coil conformation. A large increase in α-helix was induced in the presence of micelles or vesicles formed by anionic lipids. Less pronounced changes in secondary structure were induced by zwitterionic detergents but no changes were observed in the presence of neutral detergents or vesicles composed of phosphatidylcholine

Phosphatidylethanolamine mediates insertion of the catalytic domain of leader peptidase in membranes

AbstractLeader peptidase is an integral membrane protein of E. coli and it catalyses the removal of most signal peptides from translocated precursor proteins. In this study it is shown that when the transmembrane anchors are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. Furthermore, the purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment around the catalytically important residue serine 90 is required for the interaction with membranes

Theory of Lipid Polymorphism: Application to Phosphatidylethanolamine and Phosphatidylserine

We introduce a microscopic model of a lipid with a charged headgroup and flexible hydrophobic tails, a neutral solvent, and counter ions. Short-ranged interactions between hydrophilic and hydrophobic moieties are included as are the Coulomb interactions between charges. Further, we include a short-ranged interaction between charges and neutral solvent, which mimics the short-ranged, thermally averaged interaction between charges and water dipoles. We show that the model of the uncharged lipid displays the usual lyotropic phases as a function of the relative volume fraction of the headgroup. Choosing model parameters appropriate to dioleoylphosphatidylethanolamine in water, we obtain phase behavior which agrees well with experiment. Finally we choose a solvent concentration and temperature at which the uncharged lipid exhibits an inverted hexagonal phase and turn on the headgroup charge. The lipid system makes a transition from the inverted hexagonal to the lamellar phase which is related to the increased waters of hydration correlated with the increased headgroup charge via the charge-solvent interaction. The polymorphism displayed upon variation of pH mimics that of the behavior of phosphatidylserine.Comment: Submitte

Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane

This study identifies FtsW as the flippase that translocates lipid-linked peptidoglycan precursors across the cell membrane during bacterial cell wall synthesis