1993 Fiscal Year Final Research Report Summary
Structure and mechanism in cation-coupled transport systems
| Project/Area Number |
04044122
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Okayama University |
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Principal Investigator |
TSUCHIYA Tomofusa Okayama University, Faculty of Pharmaceutical Sciences, 薬学部, 教授 (80012673)
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| Co-Investigator(Kenkyū-buntansha) |
WILSON Thomas H. Harvard Medical School, 医学部, 教授
KURODA Masayuki Okayama University, Gene Research Center, 遺伝子実験施設, 助手
TSUDA Masaaki Okayama University, Faculty of Pharmaceutical Sciences, 薬学部, 助教授 (80132736)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Transport mechanism / Cation coupling / Transport protein / Transport regulation |
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| Research Abstract |
Cation/substrate symport is a major mechanism of active solute transport in cell membranes. In many cation/substrate symport systems, Na^+ or H^+ is utilized as the coupling cation. Namely, the driving force for the symport is an electrochemical potential of Na^+ or H^+. An electrochemical potential of Na^+ is established by Na^+/H^+ antiporter. In this study, we investigated structure of the transport proteins and mechanism of transport in the symporter and the antiporter.
The melibiose transport system of Escherichia coli and Salmonella Typhimurius utilizes either Na^+, H^+ or Li^+ as the coupling cation for symport depending on substrate transported. We isolated many types of mutant which showed altered cation specificity, altered substrate specificity, altered activity or altered temperature sensitivity. We cloned the mutant types melB gene, identified substitution of nucleotide and identified substituted amino acid residues in the melibiose transport protein. We found many Pro to Ser replacements. Then we constructed many mutant types of the melibiose transport protein by site-directed mutagenesis. Properties of the mutated melibiose transport proteins were analyzed. Thus, we revealed structure-function relationship and role of amino acid residues in the melibiose transport protein.
Function of the melibiose transport system is regulated by phospho-transferase system. We identified amino acid residues and domain in the melibiose transporter, which are involved in the interaction with a regulatory protein.
We also characterized Na^+/H^+ antiporter, and cloned and sequenced nhaA gene encoding the antiporter. Thus, we obtained information about structure-function relationship in the Na^+/H^+ antiporter.
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