Effect of the unsaturation of phospholipid acyl chains on leucine transport of Lactococcus lactis and membrane permeability

The effect of the degree of unsaturation of the phospholipid acyl chains on the branched-chain amino acid transport system of Lactococcus lactis was investigated by the use of a membrane fusion technique. Transport activity was analyzed in hybrid membranes composed of equimolar mixtures of synthetic unsaturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in which the number of cis double bonds in the 18-carbon acyl chains was varied. The accumulation level and initial rate of both counterflow and protonmotive-force driven transport of leucine decreased with increasing number of double bonds. The reduction in transport activity with increasing number of double bonds correlated with an increase in the passive permeability of the membranes to leucine. The membrane fluidity was hardly affected by the double bond content. It is concluded that the degree of lipid acyl chain unsaturation is a minor determinant of the activity of the branched chain amino acid transport system, but effects strongly the passive permeability of the membrane.

BACTERIAL SOLUTE TRANSPORT PROTEINS IN THEIR LIPID ENVIRONMENT

The cytoplasmic membrane of bacteria is a selective barrier that restricts entry and exit of solutes. Transport of solutes across this membrane is catalyzed by specific membrane proteins. Integral membrane proteins usually require specific lipids for optimal activity and are inhibited by other lipid species. Their activities are also sensitive to the lipid bilayer dynamics and physico-chemical state. Bacteria can adapt to changes in the environments (respective temperature, hydrostatic pressure, and pH) by altering the lipid composition of the membrane. Homeoviscous adaptation results in the maintenance of the liquid-crystalline phase through alterations in the degree of acyl chain saturation and branching, acyl chain length and the sterol content of the membrane. Homeophasic adaptation prevents the formation of non-bilayer phases, which would disrupt membrane organization and increase permeability. A balance is maintained between the lamellar phase, preferring lipids, and those that adopt a non-bilayer organization. As a result, the membrane proteins are optimally active under physiological conditions. The molecular basis of lipid-protein interactions is still obscure. Annular lipids stabilize integral membrane proteins. Stabilization occurs through electrostatic and possibly other interactions between the lipid headgroups and the charged amino acid residues close to the phospholipid-water interface, and hydrophobic interactions between the fatty acyl chains and the membrane-spanning segments. Reconstitution techniques allow manipulation of the lipid composition of the membrane in a way that is difficult to achieve in vivo. The physical characteristics of membrane lipids that affect protein-mediated transport functions have been studied in liposomal systems that separate an inner and outer compartment. The activity of most transport proteins is modulated by the bulk physical characteristics of the lipid bilayer, while specific lipid requirements appear rare

Bacterial solute transport proteins in their lipid environment

The cytoplasmic membrane of bacteria is a selective barrier that restricts entry and exit of solutes. Transport of solutes across this membrane is catalyzed by specific membrane proteins. Integral membrane proteins usually require specific lipids for optimal activity and are inhibited by other lipid species. Their activities are also sensitive to the lipid bilayer dynamics and physico-chemical state. Bacteria can adapt to changes in the environments (respective temperature, hydrostatic pressure, and pH) by altering the lipid composition of the membrane. Homeoviscous adaptation results in the maintenance of the liquid-crystalline phase through alterations in the degree of acyl chain saturation and branching, acyl chain length and the sterol content of the membrane. Homeophasic adaptation prevents the formation of non-bilayer phases, which would disrupt membrane organization and increase permeability. A balance is maintained between the lamellar phase, preferring lipids, and those that adopt a non-bilayer organization. As a result, the membrane proteins are optimally active under physiological conditions. The molecular basis of lipid-protein interactions is still obscure. Annular lipids stabilize integral membrane proteins. Stabilization occurs through electrostatic and possibly other interactions between the lipid headgroups and the charged amino acid residues close to the phospholipid-water interface, and hydrophobic interactions between the fatty acyl chains and the membrane-spanning segments. Reconstitution techniques allow manipulation of the lipid composition of the membrane in a way that is difficult to achieve in vivo. The physical characteristics of membrane lipids that affect protein-mediated transport functions have been studied in liposomal systems that separate an inner and outer compartment. The activity of most transport proteins is modulated by the bulk physical characteristics of the lipid bilayer, while specific lipid requirements appear rare.

Acidic phospholipids are required during solubilization of amino acid transport systems of Lactococcus lactis

The branched-chain amino acid transport system of Lactococcus lactis was solubilized with n-octyl β-D-glucopyranoside and reconstituted into proteoliposomes. Transport activity was recovered only when solubilization was performed in the presence of acidic phospholipids. Omission of acidic phospholipids during solubilization resulted in an inactive transport protein and the activity could not be restored in the reconstitution step. Similar results have been obtained for the arginine/ornithine exchange protein from Pseudomonas aeruginosa and L. lactis. Functional reconstitution of the transport protein requires the presence of aminophospholipids or glycolipids in the liposomes. We propose that during the detergent solubilization the acidic phospholipids protect the transport systems against denaturation by preventing delipidation

Reconstitution of the Leucine Transport System of Lactococcus lactis into Liposomes Composed of Membrane-Spanning Lipids from Sulfolobus acidocaldarius

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., Δµ~H+-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.

Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis

The effect of the phospholipid acyl chain carbon number on the activity of the branched-chain amino acid transport system of Lactococcus lactis has been investigated. Major fatty acids identified in a total lipid extract of L. lactis membranes are palmitic acid (16:0), oleic acid (18:1) and the cyclopropane-ring containing lactobacillic acid (19∇). L. lactis membrane vesicles were fused with liposomes prepared from equimolar mixtures of synthetic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with cis mono-unsaturated acyl chains. The activity of the branched-chain amino acid carrier is determined by the bulk properties of the membrane. PE acts as an activator and PC is ineffective. Counterflow and protonmotive-force driven transport of leucine is sensitive to changes in the acyl chain carbon number of both phospholipids and maximal with dioleoyl-PE/dioleoyl-PC. Above the gel to liquid-crystalline phase transition temperature of the lipid species membrane fluidity decreased with increasing acyl chain carbon number. Our data suggest that the carbon number of the acyl chains of PE and PC determine to a large extent the activity of the transport system. This might be relevant for the interaction of PE with the transport protein. Variations in the acyl chain composition of PC exert a more general effect on transport activity. The acyl chain composition of phospholipids determines the membrane thickness. We therefore propose that the degree of matching between the lipid-bilayer and the hydrophobic thickness of the branched-chain amine acid carrier is an important parameter in lipid-protein interactions.

Lipid Requirement of the Branched-Chain Amino Acid Transport System of Streptococcus cremoris

The role of the membrane lipid composition on the transport protein of branched-chain amino acids of the homofermentative lactic acid bacterium Streptococcus cremoris has been investigated. The major membrane lipid species identified in S. cremoris were acidic phospholipids (phosphatidylglycerol and cardiolipin), glycolipids, and glycerophosphoglycolipids. Phosphatidylethanolamine (PE) was completely absent. Protonmotive force-driven and counterflow transport of leucine was assayed in fused membranes of S. cremoris membrane vesicles and liposomes composed of different lipids obtained by the freeze/thaw-sonication technique. High transport activities were observed with natural S. cremoris and Escherichia coli lipids, as well as with mixtures of phosphatidylcholine (PC) with PE or phosphatidylserine. High transport activities were also observed with mixtures of PC with monogalactosyl diglyceride, digalactosyl diglyceride, or a neutral glycolipid fraction isolated from S. cremoris. PC or mixtures of PC with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activities. In mixtures of PC and methylated derivatives of PE, both counterflow and protonmotive force-driven transport activities decreased with increasing degree of methylation of PE. The decreased transport activity in membranes containing PC could be restored by refusion with PE-containing liposomes. These results demonstrate that both aminophospholipids and glycolipids can be activators of the leucine transport system from S. cremoris. It is proposed that aminophospholipids in Gram-negative bacteria and glycolipids in Gram-positive bacteria have similar functions with respect to solute transport.

The stability and functional properties of proteoliposomes mixed with dextran derivatives bearing hydrophobic anchor groups

Liposomes composed of Escherichia coli phospholipid were coated with polysaccharides bearing hydrophobic palmitoyl anchors. The effect on the stability of liposomes without or with integral membrane proteins was investigated. A high concentration of hydrophobized dextrans protected the liposomes against detergent degradation, decreased the fluidity of the membranes, prevented fusion of the liposomes and enhanced their stability. Proteoliposomes containing beef heart cytochrome-c oxidase and the lactose transport carrier of E. coli were similarly affected by coating with the dextrans. Under these conditions both membrane proteins were still active. Long-term stability of the coated liposomes was obtained only in the absence of the integral membrane proteins