A. Rietveld

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non‐bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non‐bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non‐bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non‐bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non‐bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.

Molecular aspects of the bilayer stabilization induced by poly(l-lysines) of varying size in cardiolipin liposomes

The interaction between poly(L-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and 31P- and 13C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca2+ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca2+, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca2+ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca2+, the lipid was organized in the hexagonal HII phase in the presence of Ca2+. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes.

Interaction of cytochrome c and its precursor apocytochrome c with various phospholipids.

The effects of cytochrome c and apocytochrome c on the structural properties of various membrane phospholipids in model systems were compared by binding, calorimetric, permeability, 31P n.m.r. and freeze‐fracture experiments. Both cytochrome c and apocytochrome c experience strong interactions only with negatively charged phospholipids; apocytochrome c interacted more strongly than cytochrome c. These interactions are primarily electrostatic but also have a hydrophobic character. Cytochrome c as well as apocytochrome c induces changes in the structure of cardiolipin liposomes as is shown by 31P n.m.r. and freeze‐fracture electron microscopy. Cytochrome c does not affect the bilayer structure of phosphatidylserine. In contrast, interaction of apocytochrome c with this phospholipid results in changes of the 31P n.m.r. bilayer spectrum of the liposomes and also particles are observed at the fracture faces. The results are discussed in relation to the import of the protein into the mitochondrion.

PHOSPHOLIPID STRUCTURE AND ESCHERICHIA COLI MEMBRANES

Publisher Summary This chapter discusses the current state of knowledge and indicates key experimental findings on the role individual phospholipid classes play in determining Escherichia coli membrane structure and function. Special emphasis is placed on the polymorphic behavior of the membrane lipids and the role of nonbilayer lipids. The availability of mutants in the biosynthesis of the major phospholipids has made this bacterial membrane a paradigm for understanding the relationships between lipid structure and membrane organization. Many minor membrane lipids appear to be essential in cellular signaling pathways or otherwise have a high bioactivity. Similar functions were also proposed for more abundant lipid classes. Despite these exciting developments, it remains unclear why membranes are composed of so many different bulk lipid classes. The way the lipids fulfill these and other specific functions is largely unknown.

Non-bilayer Lipids and the Inner Mitochondrial Membrane

In the last decade the fluid mosaic model (Singer and Nicholson 1972) of biological membranes has become generally accepted as it provided a rationale for many structural and functional features of membranes. More recently, however it has become increasingly clear that this model is incomplete for reasons relating to lipid composition as well as functional abilities of biological membranes. First, although the chemical variation in membrane lipids is enormous, it is surprising that most of them can be divided into only two groups on structural grounds: The lipids of the first group, including PC* and sphingomyelin, will organize themselves in bilayers when they are in the fully hydrated state (bilayer lipids). It is obvious that this property has greatly contributed to the bilayer concept of biological membranes. In contrast, the lipids in the second group do not form bilayers when they are dispersed in excess buffer (non-bilayer lipids). This group includes major lipids such as PE*, monoglucosyl and monogalactosyl diglyceride and CL4 (in the presence of Ca2+) (for review and references see Cullis and de Kruijff 1979). These lipids prefer the hexagonal HII, phase (Fig. 1). This phase consists of cylinders of lipids surrounding long aqueous channels. The unique feature of the HII phase both from a structural and functional point of view is that it allows polar lipids to be organized in a low energy configuration inside a hydrophobic environment. The reason for the abundant presence of these non-bilayer lipids in membranes is difficult to understand in terms of membrane models in which the bilayer is suggested to be the only organization available to the lipids.

A FREEZE-FRACTURE STUDY OF THE MEMBRANE MORPHOLOGY OF PHOSPHATIDYLETHANOLAMINE-DEFICIENT ESCHERICHIA COLI CELLS

Freeze-fracture electron microscopy was applied to study membrane morphology in a phosphatidylethanolamine-deficient E. coli strain. For growth, this strain requires millimolar concentrations of specific divalent cations like Mg2+ or Ca2+. These cations bring the bilayer to nonbilayer phase transition temperature of the lipids back to wild type levels by shifting the phase preference of cardiolipin in the membrane towards the inverted hexagonal (H(II)) phase. Under growth conditions, these cells show a bilayer based membrane with an intramembrane particle distribution as in wild type cells. Upon lowering the temperature, smooth areas are observed corresponding to gel state lipid bilayer domains. Ca2+ was used to manipulate the phase behavior of the membrane lipids in situ. Exposing the cells to Ca2+ up to 100 mM at 42 degrees C did not result in the appearance of nonbilayer structures, despite the fact that in total lipid extracts under these conditions the hexagonal H(II) phase was observed. However, the addition of a Ca2+ ionophore, which leads to exposure to Ca2+ of both faces of the plasma membrane, gives rise to formation of H(II) phase, stacked bilayer domains and blebbing upon addition of 50 mM CaCl2 at 42 degrees C. We conclude that the asymmetrical localization of divalent cations in the periplasm of this strain allows them to be functionally effective while membrane stability is maintained.

Interaction of the Mitochondrial Precursor Protein Apocytochrome C with Modelmembranes and its Implications for Protein Translocation

The majority of mitochondrial proteins are synthesized in the cytosol on free ribosomes in the form of precursors which are subsequently imported into the organelle by a posttranslational transport step (Hay et al., 1984). Depending on the final destination of the protein, either insertion into or translocation across one or two membranes has to occur. During this process the precursor (apoprotein) is converted into the mature holoprotein.