Purified lac permease and cytochrome o oxidase are functional as monomers.

Costello, M. Joseph; Escaig, J.; Matsushita, Kazunobu; Viitanen, Paul V.; Menick, Donald R.; Kaback, H. Ronald

doi:10.1016/s0021-9258(18)45493-2

Cited by 118 publications

(42 citation statements)

References 37 publications

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“…Because Cysl54 alone appears to be important for activity, it follows that any postulated disulphide bond formation must occur between two lac permease monomers, and dimerization must be part of the catalytic cycle. As shown (Costello et al 1987) however, the permease appears to be completely functional as a monomer. Furthermore, although Serl54 permease is defective, it retains the ability to catalyse lactose accumulation against a concentration gradient at about 10% of the rate of the wildtype molecule.…”

Section: Site-directed Mutagenesis Of Cys Residuesmentioning

confidence: 82%

“…The following considerations tend to exclude this notion: permease with Ser in place of Cysl54 catalyses lactose accumulation, albeit at 10 % of the wildtype rate. Because Ser is similar to Cys in that the hydroxyl group may mimic the sulphydryl group to an extent, Cysl54 was replaced with Val (Menick et al 1987). Permease with Vall54 catalyses transport about three times faster than permease with Ser in place of Cysl54 (i.e.…”

Section: Site-directed Mutagenesis Of Cys Residuesmentioning

confidence: 99%

“…Replacement of Arg302 with Leu, His or Lys (putative helix IX ; figure 3) yields permease with properties similar to those of permease mutated at His322, indicating that Arg302 may be involved in the pathway of H+ translocation before His322 (Menick et al 1987 In marked contrast, replacement of Ser300 with Ala (helix IX), Ser306 with Ala (helix IX) or Cys333 with Ser (helix X) has no significant effect on permease activity, thereby highlighting the specificity of Arg302, His322 and Glu325 and providing further support for the contention that single amino acid changes do not indiscriminately cause conformational alterations even within a relatively localized portion of the permease (out of approximately 70 independent mutations made thus far in the permease, about two-thirds have no effect on activity). Furthermore, by molecular modelling of putative helices IX and X, it can be shown that the guanidino group in Arg302 may be sufficiently close to His322 to participate in hydrogen bonding with the imidazole ring that, in turn, may be hydrogen-bonded to the carboxylate of Glu325 (figure 3).…”

Section: Mechanismmentioning

confidence: 99%

“…Kaback (1983Kaback ( , 1986a; Viitanen et al (1986); Wright et al (1986) for reviews). The lacY gene has been cloned and sequenced (Biichel et al 1980) and the permease has been purified to homogeneity, reconstituted into proteoliposomes and demonstrated to be solely responsible for P-galactoside transport as a monomer (Costello et al 1987). Lac permease is representative of a wide range of biological machines that transduce free energy stored in an electrochemical ion gradient into other forms of energy.…”

Section: Introductionmentioning

confidence: 99%

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Lac permease of Escherichia coli : on the path of the proton

1990

Phil. Trans. R. Soc. Lond. B

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Lactose/H+ symport in Escherichia coli is catalysed by a hydrophobic transmembrane protein encoded by the lacY gene that has been purified to homogeneity, reconstituted into proteoliposomes and shown to be completely functional as a monomer. Circular dichroic studies and hydropathy profiling of the amino-acid sequence of this 'lac' permease suggest a secondary structure in which the polypeptide consists of 12 hydrophobic segments in alpha-helical conformation that traverse the membrane in zig-zag fashion connected by shorter, hydrophilic domains with most of the charged residues and many of the residues commonly found in beta-turns. Support for certain general aspects of the model has been obtained from other biophysical studies, as well as biochemical, immunological and genetic approaches. Oligonucleotide-directed, site-specific mutagenesis is currently being utilized to probe the structure and function of the permease. Application of the technique provides an indication that Arg302 (putative helix IX), His322 (putative helix X) and Glu325 (putative helix X) may be sufficiently close to hydrogen-bond and that these residues play a critical role in lactose-coupled H+ translocation, possibly as components of a charge-relay type of mechanism. In contrast, Cys residues, which were long thought to play a central role in the mechanism of lactose/H+ symport, do not appear to be involved in either substrate binding or H+ translocation.

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Section: Site-directed Mutagenesis Of Cys Residuesmentioning

confidence: 82%

Section: Site-directed Mutagenesis Of Cys Residuesmentioning

confidence: 99%

Section: Mechanismmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Lac permease of Escherichia coli : on the path of the proton

1990

Phil. Trans. R. Soc. Lond. B

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“…Each of these cases gives an apparent contradiction to the rule as hydropathy analysis implicates six transmembrane helices (Aquila et al 1987;Runswick et al 1987). But it is thought that mitochondrial carriers may operate as physical dimers (Klingenberg et al 1980;Aquila et al 1987) whereas the bacterial (and other) carriers are presumed to function as monomers (Costello et al 1987), so that concordance with the rule is preserved even on this rather large scale. The single example available from chloroplasts also appears to follow the rule (more or less) and Pi: triose phosphate exchange is mediated by a homodimer ) whose subunit has seven hydrophobic segments (Flugge et al 1989).…”

Section: Families Of Anion Exchangementioning

confidence: 99%

Resolution and reconstitution of anion exchange reactions

Maloney

1990

Phil. Trans. R. Soc. Lond. B

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To illustrate the emerging class of anion exchange proteins in bacteria, this article discusses the biochemical and physiological properties of phosphate (Pi)-linked antiporters that accept glucose 6-phosphate (G6P) as their primary substrate. These systems have a bifunctional active site that binds a pair of negative charges, whether presented as a single divalent anion or a pair of monovalent substrates. Exchange stoichiometry therefore moves between the limits of 2:1 and 2:2 according to the ratio of mono- and divalent substrates at either membrane surface. This predicts an interesting reaction sequence in vivo because internal pH is more alkaline than external pH; one expects an asymmetric exchange as a pair of monovalent G6P anions moves against a single divalent G6P, and in this way an otherwise futile self-exchange of G6P can result in a net inward flux driven (indirectly) by the pH gradient. Despite their biochemical complexity, at a molecular level the Pi-linked antiporters resemble other secondary carriers. Indeed, the current listing of nearly two dozen such proteins suggests a structural theme in which the minimal functional unit has two sets of six transmembrane alpha helices separated by a central hydrophilic loop. Presently described examples show that this topology can derive from either a single protein or from pairs of identical subunits. The finding of this common structure makes it possible to begin building more detailed structural models that have more general implications.

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