Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes.

Carrasco, Nancy; Herzlinger, Doris; Mitchell, R. F.; Dechiara, S.; Danho, Waleed; Gabriel, T F; Kaback, H. Ronald

doi:10.1073/pnas.81.15.4672

Cited by 139 publications

(136 citation statements)

References 31 publications

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“…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)(10)(11)(12)(13)(14)(15)(16)(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).…”

supporting

confidence: 54%

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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supporting

confidence: 54%

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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“…Site-directed rabbit polyclonal antiserum against a dodecapeptide corresponding to the C terminus of LacY was prepared as described in ref. 25. Micro BCA protein determination and Supersignal West Pico Chemiluminescent substrate kits were from Pierce.…”

Section: Methodsmentioning

confidence: 99%

“…21, and samples (2 g of protein) were subjected to SDS/12% PAGE and Western blotting with a site-directed polyclonal antibody against the C terminus of LacY followed by an horseradish peroxidase-coupled anti-rabbit antibody (Amersham Biosciences, NA-934) as described in ref. 25.…”

Section: Construction Of Mutantsmentioning

confidence: 99%

Opening and closing of the periplasmic gate in lactose permease

Zhou¹,

Guan²,

Freites

et al. 2008

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

105

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X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N-and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H ؉ translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N-and C-six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants-Ile-40 3 Cys/Asn-245 3 Cys, Thr-45 3 Cys/Asn-245 3 Cys, and Ile-32 3 Cys/Asn-245 3 Cys-located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than Ϸ15 Å in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than Ϸ15 Å in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.cross-linking ͉ membrane proteins ͉ protein dynamics ͉ transport ͉ structure/function T he lactose permease of Escherichia coli (LacY) (1), a member of the major facilitator superfamily (MFS), transduces free energy in an electrochemical H ϩ gradient (⌬¯Hϩ) into a concentration gradient of galactopyranosides by coupling the downhill, stoichiometric translocation of H ϩ to the uphill accumulation of galactopyranosides. LacY can also use free energy released from downhill translocation of sugar in either direction to drive uphill translocation of H ϩ with generation of ⌬¯Hϩ, the polarity of which depends on the direction of the sugar gradient (1, 2).X-ray crystal structures of LacY (3-5) combined with a wealth of biochemical and biophysical data (2, 6 -10) have led to an alternating access mechanism for sugar translocation across the membrane. By this means, the inward-facing cavity closes with opening of an outward-facing cavity, thereby allowing alternative accessibility of the sugar-binding site to either face of the membrane. A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (11), and the ABC transporter Sav 1866 (12). The alternating access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (2, 13-16).Although it may seem intuitively obvious that a pathway must open on the periplasmic side of LacY to allow access of sugar to the binding site, insight is cloudy in this respect because all x-ray structures of LacY (3-5) are in the same inward-facing conformation with an open cytoplasmic cavity and a tightly closed periplasmic side. Nonetheless, site-directed alkylation (see refs. 7 and 10), single-molecule fluorescence resonance energy transfer (8) and double electron-electron resonance studies (9) clearly indicate that an outward-facing c...

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“…Deoxyoligonucleotides were synthesized on an Applied Biosystems 391 DNA synthesizer. Site-directed rabbit polyclonal antiserum against a dodecapeptide corresponding to the C terminus of lac permease (Carrasco et al, 1984) was prepared by BabCo (Richmond, California). Restriction endonucleases and T4 DNA ligase were from New England Biolabs (Beverly, Massachusetts).…”

Section: Methodsmentioning

confidence: 99%

“…Proteins were electroblotted to polyvinylidene difluoride membranes (Immobilon-PVDF; Millipore) and probed with a site-directed polyclonal antibody against the C terminus of lac permease (Carrasco et al, 1984).…”

Section: Membrane Preparationmentioning

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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Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes.

Cited by 139 publications

References 31 publications

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

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

Opening and closing of the periplasmic gate in lactose permease

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

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