Mutations in the lacY gene of Escherichia coli define functional organization of lactose permease.

Mieschendahl, M; Büchel, Dagmar E.; Bocklage, Hubertus; Müller‐Hill, Benno

doi:10.1073/pnas.78.12.7652

Cited by 55 publications

(17 citation statements)

References 23 publications

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“…In addition, proteolysis experiments with right-side-out and inverted vesicles, in which the lac carrier was specifically photolabeled with p-nitrophenyl a-D-galactopyranoside (NPG), demonstrate that the protein spans the bilayer and that the binding site is contained in a transmembrane segment (15). Finally, detailed kinetic studies (16,17) and radiation inactivation analysis (18), coupled with the observation that certain lac y gene, mutations are dominant (19), are consistent with the notion that ASH+ may induce a major alteration in subunit interaction (e.g., dimerization).…”

mentioning

confidence: 69%

Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.

Carrasco¹,

Tahara²,

Patel³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

113

117

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Monoclonal antibodies directed against the lac carrier protein purified' from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse. Several-clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture' supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles. Five stable clones were selected for expansion, formal cloning, and production of ascites fluid, and the antibodies secreted in vivo by each clone also were found to inhibit lactose transport. Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles. In contrast, the antibody has no effect on the generation ofthe proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-a-D-galactopyranoside. In order to achieve 50% inhibition of transport activity, a 2-to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane. Thus, the concentration ofantibody required for a given degree ofinhibition is proportional to the amount of lac carrier in the membrane. Finally, antibody-induced inhibition occurs within seconds, an observation suggesting that the epitope is accessible on the surface of the membrane.Transport of f-galactosides in Escherichia coli is catalyzed by the product of the lac y gene (1), the lac carrier protein, which translocates 13-galactosides with protons in a symport reaction (2). Accordingly, in the presence of a proton electrochemical gradient (ASH+), hydrogen ion moves down the electrochemical gradient and drives the uphill translocation of sugar (see ref. 3 for a review). The lac carrier was identified as a membranebound protein chemically in 1965 (4) and functionally in 1970 (5). Eight years later in rapid succession, the lac y gene was cloned in a recombinant plasmid, its product was amplified (6) and synthesized in vitro (7), and the sequence of the protein was deduced from the DNA sequence (8). Shortly thereafter, it was demonstrated (9) that lactose transport activity can be solubilized and reconstituted into proteoliposomes, and a highly specific photoaffinity label for the lac carrier was developed (10). Most recently, use of these techniques has led to the purification of a single protein, its identification as the product of the lac y gene, and the demonstration that it is the only polypeptide in the cytoplasmic membrane required for lactose/ proton symport (11, 12).The lac carrier protein is a 46.5 kilodalton (kDal) polypeptide chain of 417 amino acid residues of known sequence (8,11). A preliminary secondary structure model for the molecule has been formulated (unpublished data) based on the hydropathic nature of the protein along its sequence...

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mentioning

confidence: 69%

Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.

Carrasco¹,

Tahara²,

Patel³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

113

117

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“…The C-terminal tail of the permease (hydrophilic domain 13), for example, can be deleted without affecting activity (28,31,34), and alkaline phosphatase (18) or the biotinylation domain from a Klebsiella oxalacetate decarboxylase (35,36) can be fused to the C terminus with no effect on transport. Similarly, the N terminus (hydrophilic domain 1) can be deleted with little or no effect on function (37,38). Also, coexpression of independently cloned fragments of the lacY gene encoding molecules "split" in hydrophilic domains 2 (39) or 7 (39,40) yields permease with significant activity.…”

Section: Resultsmentioning

confidence: 99%

Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli.

McKenna

Hardy

Kaback

1992

Proc. Natl. Acad. Sci. U.S.A.

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The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane a-helices that traverse the membrane in zigzag fashion connected by hydrophilic domains, and a hydrophilic C terminus (hydrophilic domain 13 (Fig. 1). Evidence consistent with the general aspects of the model and demonstrating that the N terminus (hydrophilic domain 1) and the C terminus (hydrophilic domain 13) are on the cytoplasmic face of the membrane has been obtained from other spectroscopic measurements (5), chemical modification (6), limited proteolysis (7,8), and immunological studies (9-17). Finally, analysis of a large number of lac permease-alkaline phosphatase (lacY-phoA) fusions has provided exclusive support for the topological predictions of the 12-helix model (18).One of the more surprising results from site-directed mutagenesis studies on lac permease is that few of the amino acid residues in the protein appear to be mandatory for active transport. In addition to other site-directed mutants (see refs. 1-3), by using a permease construct devoid of cysteine residues (19)

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“…We would guess that the ion pair regions of the imbedded helices would face inward toward the aqueous channel of the pore or perhaps form interaction junctions either between imbedded helices of a single monomer, as in rhodopsin (11), or between monomers. In any model of pore formation, one would expect that some of our large group of S-missense mutants would be negatively dominant, as demonstrated for analogous mutations in the lac Y gene (25). We have recently transferred each these S mutations to the bacteriophage lambda genome, which has allowed us to assess the dominant or recessive characteristics of these mutations and also to determine the effect of each change on the physiology of the infective cycle.…”

Section: Discussionmentioning

confidence: 99%

Mutational analysis of bacteriophage lambda lysis gene S

Raab¹,

Neal²,

Garrett³

et al. 1986

J Bacteriol

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A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected. DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene. Thirty-three different missense alleles were generated. Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus. A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence. In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues. These regions resemble canonical membrane-spanning domains. In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm. The mutational pattern is largely consistent with the model. Charge changes within the putative imbedded regions render the protein nonfunctional. Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule. Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions. Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis. Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one. The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.The bacteriophage lambda has three genes which are directly involved in lysis: S, R, and Rz (16,30,37). The R gene product is a transglycosylase and is responsible for the destruction of the mechanical rigidity of the peptidoglycan (5). The Rz gene product is required for lysis only under conditions which are known to stabilize the outer membrane and may be involved in degradation of the covalent bonds between the outer membrane and the peptidoglycan (37). The S gene encodes a 107-codon polypeptide which is responsible for a lethal event at the cytoplasmic membrane, allowing the release of the R gene product to the periplasm for the degradation of the peptidoglycan (8,14,15). In our hands, only a single polypeptide, with an apparent molecular weight of about 10,000, is elaborated from the wild-type S gene (and not from an Sam mutant allele) in infected cells and in maxicells (3) and is associated with the inner membrane (4). The nature of the event at the cytoplasmic membrane is unclear, but a pleiotropic effect on mem...

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Mutations in the lacY gene of Escherichia coli define functional organization of lactose permease.

Cited by 55 publications

References 23 publications

Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.

Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.

Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli.

Mutational analysis of bacteriophage lambda lysis gene S

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