Lactose Carrier Protein of <i>Escherichia coli</i> Structure and Expression of Plasmids Carrying the Y Gene of the <i>lac</i> Operon

Teather, R. M.; Bramhall, John; Riede, Isolde; Wright, Joel E.; Fürst, M.; Aichele, Gabriele; Wilhelm, Ursula; Overath, Peter

doi:10.1111/j.1432-1033.1980.tb04715.x

Cited by 268 publications

(161 citation statements)

References 24 publications

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“…rspL, met-, thr-, recA, hsdM, hsdWF', laclYO+ZD"* (Y'A')] (Teather et al, 1980) harboring plasmid pT7-5llacY with given mutations was used for expression of lac permease from the lacZ promoter/operator. A cassette lacy gene (EMBL-X56095) devoid of Cys codons (C-less permease; van Iwaarden et al, 1991) containing the lacZpromoter/ operator was used for all lacy gene manipulations.…”

Section: Methodsmentioning

confidence: 99%

The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

Frillingos¹,

Ujwal²,

Sun³

et al. 1997

Protein Science

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Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VI11 and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to >70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262 4 Cys, Gly 268 + Cys, Asn 272 + Cys, Pro 280 + Cys, Asn 284 + Cys, Gly 287 + Cys, and Gly 288 + Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As a-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S , Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ. Keywords: active transport; bioenergetics; Cys modification; Cys replacementThe lactose permease of Escherichin coli, which catalyzes the coupled stoichiometric translocation of P-galactosides and H+ as a monomer, is an integral membrane protein with 12-transmembrane domains that are most probably in a-helical conformation (reviewed in . Site-directed mutagenesis of wild-type permease and Cys-scanning mutagenesis of a functional mutant devoid of Cys residues (C-less permease) have provided important insights into the structure and mechanism of the permease. Although as few as four amino acid residues [Glu 269 (helix VIII), Arg 302 (helix IX), His 322 (helix X), and Glu 325 (helix X)] appear to be critically involved in the symport mechanism, the activity of various active Cys-replacement mutants is altered by alkylation, and these mutants appear in clusters, suggesting that helical interfaces within the permease are important for turnover. Moreover, site-directed fluorescence spectroscopy (Jung et al

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Section: Methodsmentioning

confidence: 99%

The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

Frillingos¹,

Ujwal²,

Sun³

et al. 1997

Protein Science

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“…E. coli T206, T184, and plasmid pGM21 encoding wild-type lacy (Teather et al, 1980) or lacYwith site-directed mutations in single Cys codons have been described (Trumble et al, 1984;Menick et al, 1985Menick et al, , 1987aNeuhaus et al, 1985;Viitanen et al, 1985;Brooker & Wilson, 1986;Sarkar et al, 1986). E. coli T184 transformed with the appropriate plasmid was grown and induced with isopropyl 1-thio-8-D-galactopyranoside (IPTG) as described by Teather et al (1980) with the exception that methionine and threonine in the growth medium were replaced by 0 . 2 4 5 % casamino acids (Page & Rosenbusch, 1988).…”

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Exchange, efflux and substrate binding by cysteine mutants of the lactose permease of Escherichia coli

Iwaarden

Driessen²,

Lolkema³

et al. 1993

Biochemistry

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“…) and [8][9][10][11][12][13][14] C]uric acid (51.5 mCi mmol Ϫ1 ) were purchased from Moravek Biochemicals. Non-radioactive nucleobases and analogues were from Sigma.…”

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confidence: 99%

“…TOP10F' (Invitrogen) was used for initial propagation of recombinant plasmids. T184 (14) harboring pT7-5/ygfO (7) with given replacements was used for IPTG-inducible expression from the lacZ promoter/operator.…”

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confidence: 99%

Cysteine-scanning Analysis of Helices TM8, TM9a, and TM9b and Intervening Loops in the YgfO Xanthine Permease

Mermelekas

Georgopoulou

Kallis

et al. 2010

Journal of Biological Chemistry

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Bacterial and fungal members of the ubiquitous nucleobaseascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b, and TM8, we employed Cysscanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences 259 FLVVG-TIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLAD-GLVSVIASAV 314 and 342 TIAVMLVILGLFP 354 including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304, which had been studied previously. Of 67 single Cys mutants, 55 accumulate xanthine to 35-140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10 -20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (K i 79 M) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351, or Pro-354 impair activity or expression. Single Cys mutants V261C, A273C, G275C, and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC 50 15 M) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (EXXG-DXXAT) and in TM9a (GXXXDG).

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Lactose Carrier Protein of Escherichia coli Structure and Expression of Plasmids Carrying the Y Gene of the lac Operon

Cited by 268 publications

References 24 publications

The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

Exchange, efflux and substrate binding by cysteine mutants of the lactose permease of Escherichia coli

Cysteine-scanning Analysis of Helices TM8, TM9a, and TM9b and Intervening Loops in the YgfO Xanthine Permease

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