THe lactose carrier of Escherichia coli is a member of a class of membrane proteins known as cation-substrate cotransporters. These types of proteins are widely found in bacteria, fungi, plant, and animal cells. Examples of human genetic diseases are known in which the primary lesion involves a defect in a cation- substrate cotransporter. The primary aim of the proposed research is a molecular understanding of the relationship between the protein structure of the lactose carrier and its function of cotransporting H+ and lactose into the bacterial cytoplasm. The sugar recognition site will be analyzed by isolating and sequencing several novel classes of lactose carrier """"""""sugar- specificity"""""""" mutants which have alterations in their ability to recognize sugars. It is expected that most """"""""sugar-specificity"""""""" mutants will involve amino acid substitutions which are at, or close to, the sugar recognition site. Further information concerning the importance of particular amino acid side chains for sugar recognition and transport will also be provided by site-directed mutagenesis. In order to obtain a better understanding of the H+ recognition site, """"""""H+-coupling"""""""" mutants will be isolated from parental strains which have aberrant H+ coupling. Some of these mutants may involve interesting changes at, or close to, the H+ recognition site and provide important information concerning the mechanism of H+ coupling. In addition, the existence of possible ionic interactions which may be important for H+ coupling will be investigated via site- directed mutagenesis. Finally, models pertaining to the secondary structure of the lactose carrier within the membrane have relied primarily on the degree of segment hydropathicity. As an alternative, the topology of the lactose carrier within the membrane will be analyzed by isolating and sequencing a collection of lac Y/pho A fusions. Overall, this work should provide important insights into the structure/function relationships within the lactose carrier. Moreover, it is hoped that the general features which are learned about the molecular mechanism of the lactose carrier will ultimately apply to other cation-substrate cotransport systems found in bacteria, fungi, plant, and animal cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053259-12
Application #
2634791
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-12-01
Project End
1999-12-31
Budget Start
1998-01-01
Budget End
1999-12-31
Support Year
12
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Miscellaneous
Type
Schools of Arts and Sciences
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Johnson, J L; Brooker, R J (2004) Control of H+/lactose coupling by ionic interactions in the lactose permease of Escherichia coli. J Membr Biol 198:135-46
Green, Aileen L; Hrodey, Heather A; Brooker, Robert J (2003) Evidence for structural symmetry and functional asymmetry in the lactose permease of Escherichia coli. Biochemistry 42:11226-33
Patzlaff, Jason S; Zhang, Jingyan; Brooker, Robert J et al. (2002) An isotope-edited FT-IR study of a symporter, the lactose permease. Biochemistry 41:7366-72
Green, A L; Brooker, R J (2001) A face on transmembrane segment 8 of the lactose permease is important for transport activity. Biochemistry 40:12220-9
Pazdernik, N J; Matzke, E A; Jessen-Marshall, A E et al. (2000) Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K-R/K, of the lactose permease of Escherichia coli. J Membr Biol 174:31-40
Patzlaff, J S; Brooker, R J; Barry, B A (2000) A reaction-induced fourier transform-infrared spectroscopic study of the lactose permease. A transmembrane potential perturbs carboxylic acid residues. J Biol Chem 275:28695-700
Green, A L; Anderson, E J; Brooker, R J (2000) A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes. J Biol Chem 275:23240-6
Johnson, J L; Brooker, R J (1999) A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose. Implications for a proposed mechanism of H+/lactose symport. J Biol Chem 274:4074-81
Patzlaff, J S; Moeller, J A; Barry, B A et al. (1998) Fourier transform infrared analysis of purified lactose permease: a monodisperse lactose permease preparation is stably folded, alpha-helical, and highly accessible to deuterium exchange. Biochemistry 37:15363-75
Pazdernik, N J; Cain, S M; Brooker, R J (1997) An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner. J Biol Chem 272:26110-6

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