Dataset Information

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

ABSTRACT: The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane alpha-helices that traverse the membrane in zigzag fashion connected by hydrophilic domains, and a hydrophilic C terminus (hydrophilic domain 13). To test whether the hydrophilic domains are important for function, each domain was independently disrupted by insertion of two or six contiguous histidine residues, and the mutants were characterized with respect to initial rate of lactose transport and steady-state level of accumulation. Remarkably, histidine insertions into 10 out of 13 hydrophilic domains result in molecules that catalyze lactose accumulation effectively, although the initial rate of transport is compromised in certain cases. In contrast, insertions into hydrophilic domain 3, 9, or 10 cause a marked decrease in transport activity. As judged by immunoblots and [35S]methionine pulse-chase experiments, diminished activity is not due to decreased expression of the mutated permeases, defective insertion into the membrane, or increased rates of proteolysis after insertion. The results (i) suggest that most of the hydrophilic domains in the permease do not play an essential role in the transport mechanism and (ii) focus on the region of the permease containing putative helices IX and X as being particularly important for activity.

SUBMITTER: McKenna E

PROVIDER: S-EPMC50676 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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

McKenna E E Hardy D D Kaback H R HR

Proceedings of the National Academy of Sciences of the United States of America 19921201 24

The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane alpha-helices that traverse the membrane in zigzag fashion connected by hydrophilic domains, and a hydrophilic C terminus (hydrophilic domain 13). To test whether the hydrophilic domains are important for function, each domain was independently disrupted by insertion of two or six contiguous histidine residues, and the mutant ...[more]

PMID: 1465425

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Project description:Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.

Dataset Information

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

Publications

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

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