Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

Overath, Peter; Weigel, Ulrich; Neuhaus, Jean‐Marc; Soppa, Jörg; Seckler, Robert; Riede, Isolde; Bocklage, Hubertus; Müller‐Hill, Benno; Aichele, Gabriele; Wright, Joel E.

doi:10.1073/pnas.84.16.5535

Cited by 32 publications

(22 citation statements)

References 30 publications

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“…The present studies show that insertion of 6-histidine residues between Tyr-2 and Tyr-3 does not diminish IIA glc binding. In contrast, a lac permease mutant (Thr-7-Ile, Met-11-Ile) does not exhibit IIA glc binding (12,14). It should be noted that Met-11 is located in transmembrane helix I; modification of this residue might result in a change in helix packing and consequently loss of IIA glc binding.…”

Section: Some 6-his Insertion Mutants Of Lac Permease Are Defective Imentioning

confidence: 94%

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Topology of allosteric regulation of lactose permease

Seok

Sun

Kaback

et al. 1997

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

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Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIA glc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIA glc with membrane-associated lactose permease was used to characterize the binding reaction. The N-and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIA glc . Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV͞V and VI͞VII and periplasmic loop VII͞VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIA glc , indicating that these regions of lactose permease may be involved in recognition of enzyme IIA glc . Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIA glc . A topological map of the proposed interaction is presented.Bacteria use a wide range of carbon sources that are transported across the cytoplasmic membrane by a variety of specific transport systems. The phosphoenolpyruvate:sugar phosphotransferase system (PTS), a multifunctional system consisting of several protein components, is widespread in bacteria. The primary function of the PTS is the concomitant phosphorylation and translocation of a variety of sugar substrates from the medium (1, 2). In the case of glucose transport in Gram-negative bacteria, three soluble proteins (enzyme I, HPr, and enzyme IIA glc ) and one membrane-bound protein (enzyme IIB, C glc ) are required for transport. In Escherichia coli, the PTS also regulates uptake of various non-PTS sugars such as lactose, maltose, melibiose, galactose, and raffinose (inhibition of non-PTS inducer transport by PTS sugars is termed ''inducer exclusion''), as well as the phosphorylation of glycerol and the synthesis of cyclic AMP (catabolite repression) (2-6). These regulatory roles of the PTS have been proposed to be mediated by the glucosespecific enzyme IIA glc , and the currently accepted model suggests that the phosphorylation state of IIA glc determines whether or not it binds to a target protein (2).E. coli lactose permease (lac permease) is an important model for the study of membrane transport proteins. Lac permease is one of the most extensively investigated secondary active transport proteins that transduce free energy stored in an electrochemical ion gradient into a solute concentration gradient (7-9). It is a hydrophobic, polytopic membrane protein that catalyzes the coupled stoichiometric translocation of ␤-galactosides and H ϩ . All available evidence indicates strongly that lac permease is comprised of a bundle of 12 transmembrane ␣-h...

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Section: Some 6-his Insertion Mutants Of Lac Permease Are Defective Imentioning

confidence: 94%

“…Although a lac permease triple mutant in the N-terminal tail was reported to lack IIA glc binding (14), it has been suggested (15,16) that the allosteric regulation of lac permease is mediated mainly by an interaction of IIA glc with the central cytoplasmic loop.…”

mentioning

confidence: 99%

Topology of allosteric regulation of lactose permease

Seok

Sun

Kaback

et al. 1997

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

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show abstract

“…Mutations in putative Helix I of the Lacy protein have been analyzed with respect to the ability of the mutant proteins to bind and translocate galactosides [284]. The mutants G24E and P28S bind normal amounts of NPG with K~ pp values somewhat lower than that of wild-type LacY, but transport with less than 5% the wild-type activity.…”

Section: Vii-d Other Residuesmentioning

confidence: 99%

“…Not only influx but also efflux down a concentration gradient and exchange are greatly reduced. From these data and the accessibility of the galactoside binding site in rightside-out and inside out membrane vesicles, it is concluded that the mutations affect the isomerization of the ternary carrier-substrate-proton (CSH) complex [284]. Recent studies in which varying numbers of amino acids have been deleted from the aminoterminus of LacY have indicated that the first 22 amino acids, corresponding with the N-terminal half of the first putative transmembrane a-helix are not essential for transport [154].…”

Section: Vii-d Other Residuesmentioning

confidence: 99%

Secondary solute transport in bacteria

Poolman

Konings

1993

Biochimica et Biophysica Acta (BBA) - Bioenergetics

202

160

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“…Nelson et al (14) and Overath et al (17) described a lactose permease triple mutant defective in its N terminus (carrying a T-to-I mutation at position 7 [T7I], M11I, and G24R) and a partial revertant (T7I and M11I), which were reported to lack IIA Glc binding. These results suggested that the N-terminal 11 residues of LacY might be involved in IIA Glc binding.…”

mentioning

confidence: 99%