The H+:lactose carrier of Escherichia coli is a representative of a broad class of membrane proteins known as cation-substrate cotransporters. The primary aim of the proposed research is an understanding of the relationship between the protein structure of the H+:lactose carrier and its physiological function of transporting H+ and lactose across the membrane. Lactose carrier mutants will be isolated with alterations in their sugar recognition site. These """"""""sugar-specificity"""""""" mutants will be subjected to DNA sequencing in order to identify amino acid residues which are close to, or a part of, the sugar recognition site. Amino acids which are postulated to be close to the sugar recognition site will be modified by group-specific reagents to determine their proximity to that site and their role during sugar recognition and transport. With regard to the H+ recognition site, negatively charged amino acid residues will be replaced with noncharged residues via site-specific mutagenesis. The """"""""site- directed"""""""" mutants will be tested for their ability to transport H+ in order to identify negatively charged amino acids which are directly involved with H+ recognition and transport. Finally, to elucidate details about the structure of the protein, regions of the lactose carrier which project into the cytoplasm will be localized by isolating and sequencing lactose carrier mutants which are unable to bind to EnzIII, a cytoplasmically-located protein of the E. coli phosphotransferase system. Overall, this work should provide considerable information concerning the sugar recognition site, the H+ recognition site, and the structure of the protein within the membrane. Furthermore, the general features which are learned about the molecular mechanism of the H+:lactose carrier may ultimately apply to other cation-substrate cotransporters which are found not only in bacteria, but also in fungi, plant, and animal cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI024204-02
Application #
3137015
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-12-01
Project End
1989-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Arts and Sciences
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Goswitz, V C; Brooker, R J (1995) Structural features of the uniporter/symporter/antiporter superfamily. Protein Sci 4:534-7
Franco, P J; Brooker, R J (1994) Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli. J Biol Chem 269:7379-86
Goswitz, V C; Brooker, R J (1993) Isolation of lactose permease mutants which recognize arabinose. Membr Biochem 10:61-70
Olsen, S G; Greene, K M; Brooker, R J (1993) Lactose permease mutants which transport (malto)-oligosaccharides. J Bacteriol 175:6269-75
Gram, C D; Brooker, R J (1992) An analysis of the side chain requirement at position 177 within the lactose permease which confers the ability to recognize maltose. J Biol Chem 267:3841-6
Matzke, E A; Stephenson, L J; Brooker, R J (1992) Functional role of arginine 302 within the lactose permease of Escherichia coli. J Biol Chem 267:19095-100
Brooker, R J (1991) An analysis of lactose permease ""sugar specificity"" mutations which also affect the coupling between proton and lactose transport. I. Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport. J Biol Chem 266:4131-8
Eelkema, J A; O'Donnell, M A; Brooker, R J (1991) An analysis of lactose permease ""sugar specificity"" mutations which also affect the coupling between proton and lactose transport. II. Second site revertants of the thiodigalactoside-dependent proton leak by the Val177/Asn319 permease. J Biol Chem 266:4139-44
Franco, P J; Brooker, R J (1991) Evidence that the asparagine 322 mutant of the lactose permease transports protons and lactose with a normal stoichiometry and accumulates lactose against a concentration gradient. J Biol Chem 266:6693-9
Brooker, R J (1990) Characterization of the double mutant, Val-177/Asn-322, of the lactose permease. J Biol Chem 265:4155-60

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