The role of lipids in membrane insertion and translocation of bacterial proteins.

Phospholipids are essential building blocks of membranes and maintain the membrane permeability barrier of cells and organelles. They provide not only the bilayer matrix in which the functional membrane proteins reside, but they also can play direct roles in many essential cellular processes. In this review, we give an overview of the lipid involvement in protein translocation across and insertion into the Escherichia coli inner membrane. We describe the key and general roles that lipids play in these processes in conjunction with the protein components involved. We focus on the Sec-mediated insertion of leader peptidase. We describe as well the more direct roles that lipids play in insertion of the small coat proteins Pf3 and M13. Finally, we focus on the role of lipids in membrane assembly of oligomeric membrane proteins, using the potassium channel KcsA as model protein. In all cases, the anionic lipids and lipids with small headgroups play important roles in either determining the efficiency of the insertion and assembly process or contributing to the directionality of the insertion process.

Translocation of phospholipids is facilitated by a subset of membrane-spanning proteins of the bacterial cytoplasmic membrane.

The mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane alpha-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2-6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (C6-NBD)-phospholipids (Kol, M. A., de Kroon, A. I., Rijkers, D. T., Killian, J. A., and de Kruijff, B. (2001) Biochemistry 40, 10500-10506). Here the dithionite reduction assay was used to measure transbilayer equilibration of C6-NBD-phospholipids in proteoliposomes, composed of Escherichia coli phospholipids and a subset of bacterial membrane proteins. It is shown that two well characterized integral proteins of the bacterial cytoplasmic membrane, leader peptidase and the potassium channel KcsA, induce phospholipid translocation, most likely by their transmembrane domains. In contrast, the ATP-binding cassette transporter from the E. coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein from E. coli, did not facilitate flop either, demonstrating specificity of protein-mediated phospholipid translocation. The results are discussed in the light of phospholipid transport across the E. coli inner membrane.

Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry.

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K(+) channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with trifluoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid specificity. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may reflect the differences in affinity of these phospholipids for KcsA in the membrane.

Monte Carlo simulations of voltage-driven translocation of a signal sequence.

Transmembrane potentials play important but poorly understood roles in many biological processes, including signal sequence-mediated protein translocation across bacterial membranes. In this study we applied Monte Carlo techniques to simulate the way the potential acts on a signal sequence. The simulations demonstrate that in the absence of a potential the signal sequence prefers insertion in both helical hairpin and transmembrane alpha-helical conformations. However, in the presence of a potential only the transmembrane alpha-helical conformation is the state of lowest energy for the signal sequence. From these results it is concluded that the membrane potential stabilizes the transmembrane orientation of a signal sequence, explaining the membrane potential dependence of preprotein translocation.

Influence of lipids on membrane assembly and stability of the potassium channel KcsA.

Recently we observed in an in vitro system that newly synthesized KcsA assembles efficiently into a tetramer in lipid vesicles [van Dalen et al. (2002) FEBS Lett. 511, 51-58]. Here we used this system to get insight into the importance of the lipid composition for KcsA membrane association and tetramerization and we compared this to the lipid dependency of the thermo-stability of the KcsA tetramer. It was found that a large amount of phosphatidylethanolamine (>40 mol%) and a lower amount of phosphatidylglycerol (approximately 20-30 mol%) were optimal for efficient KcsA membrane association and tetramerization. Strikingly, vesicles of the abundant and commonly used membrane lipid phosphatidylcholine did not support assembly, further demonstrating the importance of membrane lipid composition for KcsA assembly. The in vitro assembled KcsA tetramer showed similar thermo-stability in biological and pure lipid membranes, demonstrating that both tetramers are alike. In addition, we show that solubilization of the membrane with detergent reduces the thermo-stability of the tetramer. The highest KcsA tetramer stability was observed in intact bilayers in the presence of anionic lipids.

Components required for membrane assembly of newly synthesized K+ channel KcsA.

An Escherichia coli in vitro transcription-translation system was used to study the components involved in the biogenesis of the homotetrameric potassium channel KcsA. We show that a functional signal recognition particle pathway is essential for tetramer formation, probably to direct correct monomer insertion in the membrane. In the absence of YidC or at reduced SecYEG levels, KcsA assembly occurs with lower efficiency. Strikingly, the highest efficiency of tetramerization was observed when transcription-translation was carried out in the presence of pure lipid vesicles, demonstrating that a phospholipid bilayer is the minimal membrane requirement to form the KcsA tetramer. It is concluded that SecYEG and YidC are not required for the formation of tetrameric KcsA in vitro.