Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.     

Interaction modes of long-chain fatty acids in dipalmitoylphosphatidylcholine bilayer membrane: contrast to mode of inhalation anesthetics

The effects of long-chain fatty acids (four saturated and two unsaturated fatty acids, one derivative) on phase transitions of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were examined in the low concentration region, and the results were compared with those for an inhalation anesthetic. The effects of all fatty acids on the pre- and main-transition temperatures of the DPPC bilayer membrane appeared in the concentration range of μM order while that of the anesthetic appeared in the mM order. The appearance modes of these ligand actions were significantly different from one another. The three differential partition coefficients of the ligands between two phases of the DPPC bilayer membrane were evaluated by applying the thermodynamic equation to the variation of the phase-transition temperatures. The DPPC bilayer membranes showed the different receptivity for the ligands; the saturated fatty acids had an affinity for gel phase whereas unsaturated fatty acids and an anesthetic had an affinity for liquid-crystalline phase to the contrary. In particular, the receptivity for the ligands in the gel phase markedly changed depending on kinds of ligands. The interaction modes between the DPPC and fatty acid molecules in the gel phase were considered from the hexagonal lattice model. The disappearance compositions of the pretransition by the fatty acids coincided with the compositions at which the membrane is all covered by the units in each of which two fatty acids molecules are regularly distributed in the hexagonal lattice in a different way, and the distribution depended on the chain length and existence of a double bond for the fatty acids. The interpretation did not hold for the case of the anesthetic at all, which proved that a number of anesthetic molecules act the surface region of the bilayer membrane nonspecifically. The present study clearly implies that DPPC bilayer membranes have high ability to recognize kinds of ligand molecules and can discriminate among them with specific interaction by the membrane states.     

Transport of fatty acids across membranes by the diffusion mechanism

In early research on fatty acid transport, passive diffusion seemed to provide an adequate explanation for movement of fatty acids through the membrane bilayer. This simple hypothesis was later challenged by the discovery of several proteins that appeared to be membrane-related fatty acid transporters. In addition, some biophysical studies suggested that fatty acids moved slowly through the simple model membranes (phospholipid bilayers), which would provide a rationale for protein-assisted transport. Furthermore, it was difficult to rationalize how fatty acids could diffuse passively across the bilayer as anions. Newer studies have shown that fatty acids are present in membranes in the un-ionized as well as the ionized form, and that the un-ionized form can cross a protein-free phospholipid bilayer quickly. This flip-flop mechanism has been validated in cells by intracellular pH measurements. The role of putative fatty acid transport proteins remains to be clarified.     

Measuring the adsorption of Fatty acids to phospholipid vesicles by multiple fluorescence probes

Fatty acids (FA) are important nutrients that the body uses to regulate the storage and use of energy resources. The predominant mechanism by which long-chain fatty acids enter cells is still debated widely as it is unclear whether long-chain fatty acids require protein transporters to catalyze their transmembrane movement. We use stopped-flow fluorescence (millisecond time resolution) with three fluorescent probes to monitor different aspects of FA binding to phospholipid vesicles. In addition to acrylodan-labeled fatty acid binding protein, a probe that detects unbound FA in equilibrium with the lipid bilayer, and cis-parinaric acid, which detects the insertion of the FA acyl chain into the membrane, we introduce fluorescein-labeled phosphatidylethanolamine as a new probe to measure the binding of FA anions to the outer membrane leaflet. We combined these three approaches with measurement of intravesicular pH to show very fast FA binding and translocation in the same experiment. We validated quantitative predictions of our flip-flop model by measuring the number of H⁺ delivered across the membrane by a single dose of FA with the probe 6-methoxy-N-(3-sulfopropyl) quinolinium. These studies provide a framework and basis for evaluation of the potential roles of proteins in binding and transport of FA in biological membranes.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl2 from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which incrased in phosphatidylethanolamine content from 39% (low Na+) to 48% (high Na+) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca2+-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na+-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca2+ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

Photoaffinity labeling of fatty acid-binding proteins involved in long chain fatty acid transport in Escherichia coli.

The photoreactive fatty acid 11-m-diazirinophenoxy-[11-3H]undecanoate was shown to be taken up specifically by the fatty acid transport system expressed in Escherichia coli grown on oleate. This photoreactive fatty acid analogue was therefore used to identify proteins involved in fatty acid uptake in E. coli. The fadL protein was labeled by the probe, confirmed to be exclusively in the outer membrane and to exhibit the heat modifiable behavior typical of outer membrane proteins. The apparent pI of the incompletely denatured form of the protein having the mobility of a 33-kDa protein was 4.6 while that of the fully denatured form was consistent with the calculated value of 5.2. The denaturation was reversible depending upon the protein to detergent ratios. The photoreactive fatty acid partitions into the outer membrane, resulting in extensive photolabeling of the lipid; a high affinity fatty acid-binding site is not apparent in total membranes labeled using free fatty acids due to this large binding capacity of the outer membrane. However, when the free fatty acid concentration was controlled by supplying it as a bovine serum albumin complex, the fadL protein exhibited saturable high affinity fatty acid binding, having an apparent Kd for the probe of 63 nM. The methods described very readily identify fatty acid-binding proteins: the fact that even when the sensitivity was increased 500-fold, no evidence was found for the presence of a fatty acid-binding protein in the inner membrane is consistent with the proposal that fatty acid permeation across the plasma membrane is not protein mediated but occurs by a simple diffusive mechanism.     

Role of caveolin-1 and cholesterol in transmembrane fatty acid movement

We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.     

The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes.

1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.     

Evidence for a mechanism by which omega-3 polyunsaturated lipids may affect membrane protein function

We have calculated the lateral pressure profile from well-converged, experimentally validated, molecular dynamics simulations of hydrated lipid bilayer membranes containing highly polyunsaturated fatty acids. The three simulations, each 30 ns in length, contain omega-3 fatty acids, omega-6 fatty acids, and a mixture of omega-3 fatty acids and cholesterol and were continued from previously published simulations that demonstrated excellent agreement with a wide variety of experimental measurements. We find that the distribution of lateral stress within the hydrophobic core of the membrane is sensitively dependent on the degree of chain unsaturation and on the presence of cholesterol. Replacing omega-3 fatty acids with omega-6 chains, or incorporating cholesterol into the membrane, shifts the repulsive lateral chain pressure away from the lipid/water interface toward the bilayer interior. This may support a previously proposed mechanism by which lipid composition may affect conformational equilibrium for integral membrane proteins.     

Without a little help from 'my' friends: direct insertion of proteins into chloroplast membranes?

The chloroplast membranes are highly regulated and biological active regions of the living plant cell, which carry numerous essential proteinaceous components. For example, in the thylakoid membrane the photosynthesis apparatus, one of the most life-relevant biological machineries, is located. How these membrane proteins are targeted to and inserted into their target membranes was one of the questions we aimed to understand in the last few years. Fifteen years ago little to nothing was known about the targeting and translocation of outer envelope proteins (G.W. Schmidt and L.M. Mishkind, Annu. Rev. Biochem. 55 (1986)). Although several protein assisted pathways for translocation of proteins across the membranes have been characterised, only recent results gave insight into how membrane proteins are inserted into the chloroplast membranes. Here we will focus on the mode of insertion of a class of proteins into the outer envelope and the thylakoid membranes, which share a unique feature: they insert apparently directly into the lipid bilayer, i.e. without the help of a proteinaceous translocation pore.     

Biochemical demonstration of the involvement of fatty acyl-CoA synthetase in fatty acid translocation across the plasma membrane

Fatty acyl-CoA synthetase, the first enzyme of the beta-oxidation pathway, has been proposed to be involved in long chain fatty acid translocation across the plasma membrane of prokaryotic and eukaryotic cells. To test this proposal, we used an in vitro system consisting of Escherichia coli inner (plasma) membrane vesicles containing differing amounts of trapped fatty acyl-CoA synthetase and its substrates CoA and ATP. This system allowed us to investigate the involvement of fatty acyl-CoA synthetase independently of other proteins that are involved in fatty acid translocation across the outer membrane and in downstream steps in beta-oxidation, because these proteins are not retained in the inner membrane vesicles. Fatty acid uptake in vesicles containing fatty acyl-CoA synthetase was dependent on the amount of exogenous ATP and CoASH trapped by freeze-thawing. The uptake of fatty acid in the presence of non-limiting amounts of ATP and CoASH was dependent on the amount of endogenous fatty acyl-CoA synthetase either retained within vesicles during isolation or trapped within vesicles after isolation by freeze-thawing. Moreover, the fatty acid taken up by the vesicles was converted to fatty acyl-CoA. These data are consistent with the proposal that fatty acyl-CoA synthetase facilitates long chain fatty acid permeation of the inner membrane by a vectorial thioesterification mechanism.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl₂ from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which increased in phosphatidylethanolamine content from 39% (low Na⁺) to 48% (high Na⁺) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca²⁺-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na⁺-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca²⁺ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

Molecular mechanisms of interaction of rabbit CAP18 with outer membranes of gram-negative bacteria.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.     

Induction of non-bilayer lipid structures by functional signal peptides.

Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae.

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

Fast Diffusion of Very Long Chain Saturated Fatty Acids across a Bilayer Membrane and Their Rapid Extraction by Cyclodextrins: IMPLICATIONS FOR ADRENOLEUKODYSTROPHY*

Abnormalities in the transport of saturated very long chain fatty acids (VLCFA; >C18:0) contribute to their toxic levels in peroxisomal disorders of fatty acid metabolism, such as adrenoleukodystrophy and adrenomyeloneuropathy. We previously showed that VLCFA desorb much slower than normal dietary fatty acids from both albumin and protein-free lipid bilayers. The important step of transbilayer movement (flip-flop) was not measured directly as a consequence of this very slow desorption from donors, and the extremely low aqueous solubility of VLCFA precludes addition of unbound VLCFA to lipid membranes. We have overcome these limitations using methyl-β-cyclodextrin to solubilize VLCFA for rapid delivery to “acceptor” phosphatidylcholine vesicles (small and large unilamellar) and to cells. VLCFA binding was monitored in real time with the fluorescent probe fluorescein-labeled phosphatidylethanolamine in the outer membrane leaflet, and entrapped pyranine was used to detect flip-flop across the membrane. The upper limit of the rate of flip-flop across the membrane was independent of temperature and media viscosity and was similar for model raft and non-raft membranes as well as living cells. We further showed that cyclodextrins can extract VLCFA rapidly (within seconds) from vesicles and cells, which have implications for the mechanism and potential alternative approaches to treat adrenoleukodystrophy. Because VLCFA diffuse through the lipid bilayer, proteins may not be required for their transport across the peroxisomal membrane.     

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.     

Surface NADH oxidase of HeLa cells lacks intrinsic membrane binding motifs

Disulfide-thiol interchange proteins with hydroquinone (NADH) oxidase activities (designated NOX for plasma membrane-associated NADH oxidases) occur as extrinsic membrane proteins associated with the plasma membrane at the outer cell surface. The cancer-associated NOX protein, designated tNOX, has been cloned. The 34-kDa plasma membrane-associated form of the protein contains no strongly hydrophobic regions and is not transmembrane. No myristoylation or phosphatidylinositol anchor motifs were discovered. Evidence for lack of involvement of a glycosylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-specific phospholipase C or with nitrous acid at low pH to release the NOX protein from the surface of HeLa cells or from plasma membranes isolated from HeLa cells. Binding of NOX protein to the plasma membrane via amino acid side chain modification or by attachment of fatty acids also is unlikely based on use of specific fatty acid antisera to protein bound fatty acids and as a result of binding to the cancer cell surface of a truncated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37 degrees C for 1 h, was sufficient to release tNOX from the HeLa cell surface. Release was unaffected by protease inhibitors or divalent ions and was not accelerated by addition of cathepsin D. The findings suggest dissociable receptor binding as a possible basis for their plasma membrane association.     

Post-translational modifications of mitochondrial outer membrane proteins

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.