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P Overath

Publications and source records attributed to P Overath.

At least 91 records · Page 5Linked to original sources
PubMed1985

Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution.

The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.

Amino Acid Sequence↗
PubMed1984

Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport.

The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre-extraction of the membranes with 5-sulfosalicylate, solubilization in dodecyl-O-beta-D-maltoside, Ecteola-column chromatography, and removal of residual impurities by anti-impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles. Purification was monitored by tracer N-[3H]ethylmaleimide-labeled carrier and by binding of the substrate p-nitrophenyl-alpha-D-galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria. Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain. Large unilamellar proteoliposomes (1-5-micron diameter) were prepared from initially small reconstituted vesicles by freeze-thaw cycles and low-speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (delta mu H+) or one of its components (delta psi or delta pH). Comparison of the steady-state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport. The half-saturation constants for active uptake of lactose (KT = 200 microM) or beta-D-galactosyl-1-thio-beta-D-galactoside (KT = 50-80 microM) by the purified carrier were found to be similar to be similar to those measured in cells or cytoplasmic membrane vesicles. The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat = 3-4 s-1 for lactose) but considerably smaller than in cells (kcat = 40-60 s-1). Possible reasons for this discrepancy are discussed.

Bacterial Proteins↗
PubMed1983

Characterization of beta-galactosidase--lactose-permease chimaeras of Escherichia coli.

Escherichia coli strains have been isolated in which 3, 39 or 805 5'-end codons of lacZ, the gene for the cytoplasmic enzyme beta-galactosidase are fused to codon 9 of lacY, the gene for lactose permease. Lactose-permease-deficient cells, carrying the lacZ-Y fusions on F' lac pro episomes, are phenotypically positive on eosin/methylene blue/lactose or on melibiose plates, demonstrating that the beta-galactosidase--lactose-permease chimaeras transport lactose and melibiose in vivo. The apparent affinity for beta-D-galactopypanosyl 1-thio-beta-D-galactopyranoside (GalSGal) in cells is similar to that of the wild-type gene product. The maximum velocity of active GalSGal transport is reduced in all three fusion strains. Both lactose and p-nitrophenyl alpha-D-galactopyranoside inhibit GalSGal uptake. As demonstrated by immunoblot experiments the chimaeras cross-react with polyclonal antibodies directed against native lactose permease and they are present in the cell envelope fraction of homogenates. Their apparent molecular weights upon electrophoresis in NaDodSO4/polyacrylamide gels correspond to those expected from their respective primary sequences, taking into account the migration properties of wild-type lactose permease. It is proposed that substitution of eight N-terminal lactose permease residues by N-terminal beta-galactosidase residues neither prevents membrane incorporation of permease nor completely impairs the ability to transport galactosides actively. Alternative interpretations of the experimental results are discussed.

Biological Transport, Active↗
PubMed1983

Does the lactose carrier of Escherichia coli function as a monomer?

The purified lactose carrier of Escherichia coli (product of the lac Y gene) is shown to be a monomer in detergent micelles of dodecyl-O-beta-D-maltoside. The negative-dominant phenotype of mutant carriers (lacY-d mutants) could not be verified by measurements of the rate of galactoside transport in lacY+/Y-d diploid strains. It is proposed that the membrane-embedded carrier functions as a monomer in galactoside-H+ symport.

Biological Transport↗
PubMed1983

Peptide-specific antibody locates the COOH terminus of the lactose carrier of Escherichia coli on the cytoplasmic side of the plasma membrane.

The carboxyl-terminal decapeptide NH2-Leu-Leu-Arg-Arg-Gln-Val-Asn-Glu-Val-Ala-OH of the lactose carrier protein, the product of the lac Y gene of Escherichia coli, was synthesized, and specific anti-peptide antibodies were raised in rabbits. These antibodies bind to membrane-bound lactose carrier showing that the carboxyl terminus is accessible from the aqueous phase. The antibodies bind only to the surface of inverted cytoplasmic membrane vesicles (but not to closed, right-side-out membrane vesicles), demonstrating that the carboxyl terminus of the carrier protein is directed towards the cytoplasmic side of the plasma membrane in cells. The carboxyl terminus is a potent immunogenic epitope on the purified, detergent-solubilized carrier. Binding of peptide-specific antibodies to the carrier protein inhibits neither substrate binding nor translocation.

Amino Acid Sequence↗
PubMed1983

Repression of glycoprotein synthesis and release of surface coat during transformation of Trypanosoma brucei.

The biosynthesis of the variant surface glycoprotein (VSG) and its release from the surface of Trypanosoma brucei 427 variant clone MITat 1.4 (117) during in vitro transformation of bloodstream trypomastigotes to procyclic trypomastigotes was investigated. After transfer to the transformation medium at 27 degrees C, VSG synthesis is repressed with a half-time, t1/2 = 30 min. Concomitantly VSG-specific mRNA is lost suggesting that repression operates at the transcriptional level. The expression-linked extra gene copy, which codes for VSG, is retained during and after completion of transformation. After repression of VSG synthesis, surface VSG is shed from the cells into the culture medium. During release part of VSG (apparent mol. wt. 61 000) is proteolytically cleaved to a product (apparent mol. wt. 51 000) which represents the N-terminal domain of the protein as judged by the absence of the carbohydrate moiety normally linked to the C terminus.

Animals↗
PubMed1982

Lactose carrier protein of Escherichia coli. Reconstitution of galactoside binding and countertransport.

A procedure for the reconstitution of the lactose carrier protein, a galactoside:proton symporter in Escherichia coli, is described. Starting from cytoplasmic membranes derived from carrier-overproducing strains, essentially all proteins including 89% of the carrier are solubilized by a mixture of dodecyl/tetradecyl polyoxyethylene (n = 9.5) ether and dodecyl O-beta-D-maltoside. In the micellar state the carrier binds substrates with reduced affinity. Addition of E. coli phospholipids and removal of detergents by a hydrophobic column yields small vesicles (50-60-nm diameter). In these vesicles, about 70% of the carrier is recovered and reconstituted carrier is identical to native carrier in terms of substrate binding. After fusion of the small vesicles into larger vesicles (1-5 micrometers), rapid countertransport of galactosides is demonstrated. Attempts to show active galactoside transport by the imposition of artificial electrical potential or pH gradients were unsuccessful, most likely because the reconstituted vesicles are in fact highly permeable to protons.

Bacterial Proteins↗
PubMed1981

Function of phospholipids in Escherichia coli. Influence of changes in polar head group composition on the lipid phase transition and characterization of a mutant containing only saturated phospholipid acyl chains.

The cls mutation conferring a defect in cardiolipin synthesis (Pluschke, G., Hirota, Y., and Overath, P. (1978) J. Biol. Chem. 253, 5048-5055) has been introduced into an Escherichia coli strain defective in unsaturated fatty acid synthesis in order to study the effect of changes in polar head group composition on the ordered in equilibrium fluid phase transition of the membrane phospholipids. The defect in cardiolipin formation is compensated by an increase in phosphatidylglycerol content, resulting in a decrease of the midpoint of the phase transition by 6 degrees C. Starvation of the cls mutant strain for the unsaturated fatty acid supplement leads to the incorporation of saturated acyl chains of reduced average length into the phospholipids, and growth is inhibited although the membrane remains in a fluid state. A revertant of this strain is described which retains the parental fabB-, fadE-, and cls markers and grows in the absence of an unsaturated fatty acid supplement. A cls+ derivative of the revertant can multiply in a restricted temperature range (35-43 degrees C). It contains only saturated phospholipid acyl chains of an anomalously short average length of 14 carbon atoms but has the same polar head group composition as wild type E. coli. The results demonstrate that, under defined conditions, saturated acyl chains of reduced length are functionally equivalent to unsaturated chains.

Cardiolipins↗
PubMed1981

Structure of Escherichia coli membranes. Glycerol auxotrophs as a tool for the analysis of the phospholipid head-group region by deuterium magentic resonance.

Glycerol selectively deuterated at various positions was synthesized and supplied to the growth medium of Escherichia coli strain T131 GP, which is defective in endogenous glycerol synthesis as well as glycerol degradation and lacks the ability to synthesize cardiolipin. The procedure enables the stereospecific labeling of the membrane phospholipids (approximately 80% phosphatidylethanolamine, approximately 20% phosphatdylglycerol). Deuterium magnetic resonance spectra were obtained for cell membranes and lipid dispersions either from total lipid extractions or from purified phosphitidylglycerol or -ethanolamine. When glycerol deuterated at various positions was used, all resonances of the phospholipid glycerol backbone and the terminal glycerol moiety in phosphatidylglycerol could be assigned. The results indicate that the molecular conformation of the glycerol backbone is independent of the phospholipid species investigated and is also not altered by the presence of high amounts of membrane proteins. For the quantitative interpretation of the deuterium magnetic resonance splittings, a model is proposed which assumes essentially free rotation around the glycerol C(2)-C(3) bond combined with an asymmetric and restricted jump process around the C(1)-C(2) bond. This model is compatible with known X-ray structures of phospholipids molecules. The two deuterons of both the glycerol backbone C(1) and C(3) segments were found to be magnetically inequivalent. Stereoselective monodeuteration eliminated one set of quadrupole splittings in both cases.

Cell Membrane↗
PubMed1980

In vitro and in vivo products of E. coli lactose permease gene are identical.

The lacY gene product synthesised in vitro is identical to lactose permease isolated from cytoplasmic membranes as determined by apparent molecular weight and N-terminal amino acid sequence. The amino acid composition of the in vivo product agrees well with that predicted from the DNA sequence. The data assign the translational start on the DNA sequence and demonstrate that this protein is processed only by deformylation but not by proteolytic cleavage at the N-terminus.

Amino Acid Sequence↗
PubMed1980

Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon.

The previously described hybrid plasmid pC7 which carries lacI+O+delta(Z)Y+A+ on a 12.3 X 10(6)-Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239-248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to d(A-A-T-T) and ligated to the vector pB322. lac Y-carrying inserts of various sized (Mr 1.5-4.7 X 10(6)) were obtained. Hybrid plasmid pTE18 (2300-base-pair insert) carries part of the I (repressor) gene, the promotor-operator region, part of the Z (beta-galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18-harbouring strains the Y-gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12-16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.

Bacterial Proteins↗