Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli

Synthesis of the high-affinity K⁺-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE. K⁺ limitation or a sudden increase in osmolarity induces the expression of kdpFABC. Due to the importance of K⁺ to maintain turgor, it has been proposed that KdpD is a turgor sensor. Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates. Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E. coli mutants defective in phospholipid biosynthesis. Surprisingly, neither the lack of the major E. coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly. However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role. These results indicate that electrostatic interactions are important for the activity of KdpD. Received: 29 March 1999 / Accepted: 26 July 1999     

MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.     

Molecular chaperones and protein translocation across the Escherichia coli inner membrane

Proteins that are able to translocate across biological membranes assume a loosely folded structure. In this review it is suggested that the loosely folded structure, referred to here as the 'pre-folded conformation', is a particular structure that interacts favourably with components of the export apparatus. Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre-folded conformation and have been termed 'molecular chaperones'. Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins.     

Energy requirements for protein translocation across the Escherichia coli inner membrane

Both ATP and an electrochemical potential play roles in translocating proteins across the inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.     

Indole transport across Escherichia coli membranes

Indole has many, diverse roles in bacterial signaling. It regulates the transition from exponential to stationary phase, it is involved in the control of plasmid stability, and it influences biofilm formation, virulence, and stress responses (including antibiotic resistance). Its role is not restricted to bacteria, and recently it has been shown to include mutually beneficial signaling between enteric bacteria and their mammalian hosts. In many respects indole behaves like the signaling component of a quorum-sensing system. Indole synthesized within the producer bacterium is exported into the surroundings where its accumulation is detected by sensitive cells. A view often repeated in the literature is that in Escherichia coli the AcrEF-TolC and Mtr transporter proteins are involved in the export and import, respectively, of indole. However, the evidence for their involvement is indirect, and it has been known for a long time that indole can pass directly through a lipid bilayer. We have combined in vivo and in vitro approaches to examine the relative importance of protein-mediated transport and direct passage across the E. coli membrane. We conclude that the movement of indole across the E. coli membrane under normal physiological conditions is independent of AcrEF-TolC and Mtr. Furthermore, direct observation of individual liposomes shows that indole can rapidly cross an E. coli lipid membrane without the aid of any proteinaceous transporter. These observations not only enhance our understanding of indole signaling in bacteria but also provide a simple explanation for the ability of indole to signal between biological kingdoms.     

SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane

Recent insight into the biochemical mechanism of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein-SecB complex, binding to inner-membrane anionic phospholipids, insertion into the membrane biiayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate-determining step in protein secretion, SecA synthesis is co-ordinated with the activity of the protein export pathway. This form of negative reguiation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA-dependent catalysis of protein export and the details of secA regulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways.     

Negatively charged phospholipids suppress IFN-gamma production in T cells

The effect of phospholipids on IFN-gamma production in mouse T cells was investigated. Phosphatidylserine (PS), which has a negatively charged head group, completely inhibited IFN-gamma production in splenic na?ve T cells and antigen-dependent IFN-gamma production in Th1 clone 42-6A cells, whereas other phospholipids, which have neutrally charged head group, had no effect. The structural requirements for IFN-gamma inhibitory effects by PS were investigated, and dimyristoyl-PS (C14: 0) and dipalmitoyl-PS (C16: 0) had no effect on IFN-gamma production, and interestingly, distearoyl-PS (18: 0) increased IFN-gamma production. Dioleoyl-PS (C18: 1), dilinoleoyl-PS (C18: 2), and oleoyl-lyso-PS (C18: 1) completely inhibited IFN-gamma production. To clarify this mechanism, we focused on the stability of IFN-gamma mRNA, and the treatment of splenic na?ve T cells with PS brought about 40% reductions in IFN-gamma mRNA expression in the presence of actinomycin D. Collectively, IFN-gamma inhibitory effects by PS are highly dependent on the molecular structure of PS and involve the decreasing of the stability of IFN-gamma mRNA.     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.     

Sequential translocation of an Escherchia coli two‐partner secretion pathway exoprotein across the inner and outer membranes

In Gram‐negative bacteria, a variety of high molecular weight ‘exoproteins’ are translocated across the outer membrane (OM) via the two‐partner secretion (TPS) pathway by interacting with a dedicated transporter. It is unclear, however, whether the translocation of exoproteins across the OM is coupled to their translocation across the inner membrane (IM). To address this question, we separated the production of an Escherichia coli O157:H7 exoprotein (OtpA) and its transporter (OtpB) temporally by placing otpA and otpB under the control of distinct regulatable promoters. We found that when both full‐length and truncated forms of OtpA were expressed prior to OtpB, a significant fraction of the exoprotein was secreted. The results indicate that OtpA can be translocated into the periplasm and briefly remain secretion‐competent. Furthermore, by engineering cysteine residues into OtpA and using disulphide bond formation as a reporter of periplasmic localization, we obtained additional evidence that the C‐terminus of OtpA enters the periplasm before the N‐terminus is translocated across the OM even when OtpA and OtpB are expressed simultaneously. Taken together, our results demonstrate that the translocation of a TPS exoprotein across the OM can occur independently from its translocation across the IM.     

Negatively charged phospholipids and their position in the cholesterol affinity sequence

The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyelin with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin.From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin ? phosphatidylserine, phosphatidylglycerol > phosphatidylcholine ? phosphatidylethanolamine.     

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.     

Carbon source-dependent synthesis of SecB, a cytosolic chaperone involved in protein translocation across Escherichia coli membranes.

SecB is a cytosolic chaperone involved in protein translocation across cytoplasmic membranes in Escherichia coli. It has been shown to be required for efficient translocation of a subset of precursor proteins but is not essential for cell viability. This study investigated whether synthesis of SecB is growth rate dependent. Interestingly, the total amount of SecB synthesized in the cells was relatively small. Moreover, the levels of SecB were found to be carbon source dependent since more SecB was produced in cells grown in glycerol media than in cells grown in glucose media, regardless of the growth rate. This is in contrast to the other Sec proteins, whose synthesis is growth rate dependent and not related to glucose as a carbon source. In addition, cyclic AMP (cAMP) partially relieves the lower levels of SecB observed in glucose medium, a compensatory effect that depends on the presence of both cya and crp gene products. Thus, the glucose-dependent synthesis of SecB may be related to the cAMP-cAMP receptor protein complex-mediated activation.     

Protein translocation across chloroplast envelope membranes

Nuclear-encoded chloroplast proteins are imported from the cytosol into the chloroplast stroma by a common translocation machinery. Several components of the import apparatus, including GTP-binding proteins and Hsp70 proteins, have recently been identified and characterized. This review discusses the role of these proteins in chloroplast protein import.     

Protein translocation across mitochondrial membranes.

Protein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes. In particular we will focus on recently identified components of the mitochondrial import apparatus.     

Functions in outer and inner membranes of Escherichia coli for ferrichrome transport.

Mutants of the fhuA gene of Escherichia coli K-12, which encodes a receptor protein in the outer membrane, took up ferrichrome after exposure to pronase, whereas fhuB mutants remained transport negative. The latter finding supports our previous proposal that fhuB mutants are defective in a function that residues in the cytoplasmic membrane. Cells remained completely viable after treatment with pronase, although they became sensitive to the antibiotic actinomycin.