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W Dowhan

Publications and source records attributed to W Dowhan.

At least 55 records · Page 3Linked to original sources
PubMed1993

Alterations in the electron transfer chain in mutant strains of Escherichia coli lacking phosphatidylethanolamine.

Inside-out sealed membrane vesicles were prepared from strains of Escherichia coli engineered to be lacking in the major phospholipid of this organism, phosphatidylethanolamine (DeChavigny, A., Heacock, P. N., and Dowhan, W. (1991) J. Biol. Chem. 266, 5323-5332). The energy transducing properties, namely the ability to generate a proton gradient directed inward and to transport electrons to molecular oxygen, were compared to those of membranes isolated from wild type cells containing normal levels of phosphatidylethanolamine. Membranes from both cell types were equal in their ability to oxidize succinate and lactate as well as hydrolyze ATP with the generation of proton gradients of similar magnitude, thus establishing the structural integrity of the membrane barrier and basic functionality of the energy transducing systems in the mutant membranes. However, mutant membranes were reduced by about 80% in their type II NADH dehydrogenase-dependent oxidase activity which resulted in a reduced ability to generate a proton gradient using NADH as an energy source. Use of artificial electron acceptors indicated that the level of type II NADH dehydrogenase activity was normal. Whole chain NADH oxidase activity could be restored by addition of short chain analogs of the naturally occurring Q8, even though the level of the Q8 pool in both cell types was the same. These results suggest that the function of Q8 in linking type II NADH dehydrogenase with the terminal oxidase(s) is dependent on the phosphatidylethanolamine content of the surrounding phospholipid matrix.

Cell Membrane↗
PubMed1993

Polymorphic regulation of membrane phospholipid composition in Escherichia coli.

To investigate whether the phase preference of Escherichia coli membrane lipids is regulated by adjustment of the ratio of bilayer to non-bilayer lipids, the lipid biosynthetic mutant AD93 was used. This strain lacks the ability to synthesize phosphatidylethanolamine, a non-bilayer lipid which normally accounts for 70-80% of the phospholipids in the E. coli membrane. The lack of phosphatidylethanolamine is compensated by a large increase in the levels of phosphatidylglycerol and cardiolipin. This strain has an absolute requirement for high concentrations of divalent cations. Since divalent cations are known to induce HII phase formation in cardiolipin model systems, this suggests that cardiolipin in combination with divalent cations replaces phosphatidylethanolamine in the membrane and that it is the non-bilayer forming property of the latter which is important for membrane functioning. The growth of the mutant strain was found to be dependent on the type and concentration of divalent cations present in the growth medium. In media supplemented with MgCl2 and CaCl2, growth was maximal at concentrations of 30-50 mM. In the presence of SrCl2 a growth optimum was observed at 15 mM. The cells did not grow in the presence of BaCl2 or at high concentrations of SrCl2. Furthermore, this strain was found to adapt the lipid composition of the membrane in reaction to the type of cation present during growth. The phase behavior of dispersions of mutant and parental E. coli derived lipids was studied under a variety of conditions, using 31P NMR. In the presence of the growth-promoting cations at concentrations where there is maximal growth, a bilayer to non-bilayer transition was observed in the lipid dispersions in a narrow temperature range, approximately 10 degrees C above the growth temperature, whereas at concentrations where there is no growth, a bilayer structure was observed at all temperatures tested. This suggests a polymorphic regulation of membrane lipid composition in order to maintain a propensity toward type II structure formation in the membrane.

Calcium Chloride↗
PubMed1993

Role of acidic lipids in the translocation and channel activity of colicins A and N in Escherichia coli cells.

Colicins A and N are pore-forming bacterial toxins that kill Escherichia coli cells. Their mode of action involves three steps; binding to specific receptors located in the outer membrane, translocation through this membrane and the periplasm, and channel formation in the inner membrane. In-vitro studies have shown that negatively charged phospholipids are an absolute requirement for the channel formation of colicin A. Using HDL11 strain, in which the phosphatidylglycerol (PtdGro) content was altered by varying the synthesis of the PtdGro-phosphate synthase, the effect of envelope PtdGro content on the activity of colicin A was studied in vivo. The formation by colicin A of a voltage-gated channel in the cytoplasmic membrane results in an efflux of cytoplasmic potassium. This efflux is preceded by a lag time which is related to the time needed by the toxin to cross the cell envelope. This lag time is higher when the cells have a reduced PtdGro level, suggesting that the receptor/translocation machinery of colicin A (OmpF, BtuB and Tol QRAB proteins) is altered in the absence of PtdGro. The rate of potassium efflux is also greatly reduced when the PtdGro content is decreased, suggesting that a certain level of PtdGro is indeed required for proper insertion of the colicin-A channel. In contrast, the activity of colicin N does not show any PtdGro dependence. The difference between the behavior of colicin A and that of colicin N is discussed.

Biological Transport↗
PubMed1993

The pss and psd genes are required for motility and chemotaxis in Escherichia coli.

Mutants of Escherichia coli defective in phosphatidylserine synthase (encoded by pss) and phosphatidylserine decarboxylase (encoded by psd) make cell membranes deficient in phosphatidylethanolamine. In this report we show that wild-type pss and psd genes are required for motility and chemotaxis. Null mutants or strains with temperature-sensitive pss or psd mutations grown at high temperature (35 degrees C) were nonmotile. They lacked flagella and showed reduced rates of transcription of the flhD master operon (encoding FlhD and FlhC), the fliA operon (encoding sigma F), and the fliC operon (encoding flagellin). At low temperature (25 degrees C), the temperature-sensitive mutant cells showed motility and chemotaxis but at reduced levels. The extent of the motility and chemotaxis defects in the mutants was correlated with the amount of phosphatidylethanolamine in the membranes, suggesting a link between membrane phospholipid composition and expression of the flagellum chemotaxis regulon.

Bacterial Proteins↗
PubMed1992

The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified.

Escherichia coli↗
PubMed1991

Phosphatidylethanolamine is not essential for the N-acylation of apolipoprotein in Escherichia coli.

It has been postulated that the N-acyl fatty acid attached to the amino terminus of the major Escherichia coli lipoprotein is derived from the fatty acid at the 1-position of phosphatidylethanolamine (PtdEtn) (Jackowski, S., and Rock, C.O. (1986) J. Biol. Chem. 261, 11328-11333). To ascertain the role of PtdEtn in the conversion of apolipoprotein to the mature lipoprotein, the lipoprotein from E. coli strain AH930 (pss::kan) containing a null mutation in the phosphatidylserine synthase gene (pss) was studied. Pulse labeling with [35S]methionine for 30 s or 5 min revealed the formation of mature lipoprotein in both wild-type (W3110) and mutant (AH930) cells. [3H]Palmitate-labeled lipoproteins from both the mutant and wild-type cells were found to contain nearly identical amounts of alkali-resistant (amide-linked, 41-42%) and alkali-labile (ester-linked, 58-59%) fatty acids. Edman degradation and dansylation of the immuno-affinity-purified [35S]cysteine-labeled lipoprotein showed that the NH2 terminus of the lipoprotein in the mutant was blocked as in the wild type. In vitro assay of apolipoprotein N-acyltransferase using membranes either from the mutant or the wild-type strain as the source of both the enzyme and the acyl donor revealed that both membranes were equally active in the conversion of [35S]methionine-labeled apolipoprotein to lipoprotein. These data strongly suggest that PtdEtn is not essential for the N-acylation of apolipoprotein to form lipoprotein, and other major phospholipids such as phosphatidylglycerol and cardiolipin can serve as the donor of fatty acid in the N-acylation of apolipoprotein.

Acylation↗
PubMed1991

Negatively charged phospholipids restore prePhoE translocation across phosphatidylglycerol-depleted Escherichia coli inner membranes.

Translocation of outer membrane precursor proteins across the Escherichia coli inner membrane is severely hampered in lipid biosynthetic mutants with strongly reduced phosphatidylglycerol (PG) levels (De Vrije, T., De Swart, R. L., Dowhan, W., Tommassen, J., and De Kruijff, B. (1988) Nature 334, 173-175; Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271-280). Two independent methods were used to demonstrate that anionic lipids by virtue of their negative head-group charge are involved in membrane translocation of the precursor of the pore protein PhoE. Using a lipid transfer protein-based method we show that introduction from lipid vesicles of PG and other acidic phospholipids but not of phosphatidylcholine restores efficient translocation across the membrane of PG-depleted inner membrane vesicles. Moreover, translocation was found to be proportional to the PG content in vesicles isolated from strain HDL11 in which the PG content was altered by varying the synthesis of the PG-phosphate synthase.

Bacterial Outer Membrane Proteins↗
PubMed1991

Sequence and inactivation of the pss gene of Escherichia coli. Phosphatidylethanolamine may not be essential for cell viability.

Phosphatidylethanolamine is the only zwitterionic phospholipid in Escherichia coli and accounts for 70-80% of the total glycerophospholipids of this organism. To investigate the function of phosphatidylethanolamine in E. coli, we constructed an inactivated allele (pss93::kan) of the gene encoding the phosphatidylserine synthase which catalyzes the committed step to the synthesis of phosphatidylethanolamine. Growth of this mutant was dependent on a plasmid-borne copy of the wild type gene. After curing the mutant of the wild type gene, growth stopped when the content of phosphatidylethanolamine reached 30% of the total phospholipid. Divalent metal ions at millimolar concentrations suppressed the growth phenotype of the mutant in the following order of efficiency: Ca2+ greater than Mg2+ greater than Sr2+. Although phosphatidylserine synthase activity was not detectable, phosphatidylethanolamine was still present at 0.007% of the total phospholipid after growth for many generations in rich medium containing 20 mM Mg2+. The remainder of the phospholipid was primarily phosphatidylglycerol and cardiolipin with no other unique phosphate-containing chloroform-soluble material present. The phospholipid to protein ratio and the fatty acid composition were very similar to the parental strain. The broad divalent metal ion auxotrophy brought about by the lack of phosphatidylethanolamine suggests a primarily structural role for this phospholipid in E. coli.

Amino Acid Sequence↗
PubMed1991

Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein.

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.

Carrier Proteins↗
PubMed1991

Regulation of eukaryotic phospholipid metabolism.

Phospholipids have diverse and critical roles in cellular metabolism and function. Questions about the mechanisms of regulation of phospholipid synthesis are being investigated with a variety of systems and approaches. For example, the yeast Saccharomyces cerevisiae is an organism in which both biochemical and genetic analyses are used. Biochemical approaches have yielded considerable information on the regulatory properties of enzymes of phospholipid biosynthesis. Studies of the activity of purified phosphatidylserine synthase have suggested how that enzyme is influenced by membrane phospholipids in the cell. The enzyme that regulates mammalian phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, is also influenced by phospholipids. In addition, the activity of this enzyme often correlates with its translocation to membranes. The location of such enzymes in the cell is of particular interest in light of the possibility that the enzymatic reactions may be efficiently coupled in vivo. Techniques to render cultured cells permeable to phosphorylated molecules indicated that the enzymes of phosphatidylcholine biosynthesis may exist in an organized compartment so that the precursors of phosphatidylcholine are efficiently channeled through the pathway. To ask how phospholipids are transported in the cell, a combined biochemical and genetic approach has been used. These studies have revealed that the phosphatidylinositol/phosphatidylcholine transfer protein, considered to mediate intracellular phospholipid transfer, is a critical component of the secretory pathway for proteins. These results have allowed formulation of a number of new questions on the regulation of phospholipid metabolism and its relationship to general membrane processes.

Animals↗
PubMed1990

An essential role for a phospholipid transfer protein in yeast Golgi function.

Progression of proteins through the secretory pathway of eukaryotic cells involves a continuous rearrangement of macromolecular structures made up of proteins and phospholipids. The protein SEC14p is essential for transport of proteins from the yeast Golgi complex. Independent characterization of the SEC14 gene and the PIT1 gene, which encodes a phosphatidylinositol/phosphatidylcholine transfer protein in yeast, indicated that these two genes are identical. Phospholipid transfer proteins are a class of cytosolic proteins that are ubiquitous among eukaryotic cells and are distinguished by their ability to catalyse the exchange of phospholipids between membranes in vitro. We show here that the SEC14 and PIT1 genes are indeed identical and that the growth phenotype of a sec14-1ts mutant extends to the inability of its transfer protein to effect phospholipid transfer in vitro. These results therefore establish for the first time an in vivo function for a phospholipid transfer protein, namely a role in the compartment-specific stimulation of protein secretion.

Carrier Proteins↗