Secondary active transport systems of amino acids in E. coli

What is secondary active transport system?

Transport proteins work in many places in a cell to accumulate or passively transport ions, sugars, drugs, amino acids and so on. Secondary active transport system is an active transport system of a solute driven by an electrochemical gradient of other solute (proton, sodium ion, etc). These transport proteins play important roles in cell metabolism. But the three dimensional structures are still unknown, because they are very hydrophobic and difficult to be purified (thus, to be crystallized).

We have been studying proline and glutamate transport proteins (and their genes) in E. coli. We can amplify and purify them by using cloned genes. Now we expect that our biochemical and structural studies of these well-characterized proteins should give fundamental and universal insights into the enzymological and energy coupling mechanisms of secondary active transport systems.

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What we have done

• Cloning and sequencing of proline and glutamate carrier genes
  　　Motojjima et al., Proc. Natl. Acad. Sci. USA 76, 6255-6259 (1979)
  　　Nakao et al., Mol. Gen. Genet. 208, 70-75 (1987)
  　　Deguchi et al., J. Biol. Chem. 265, 21704-21708 (1990)
• Regulation of gene expression of the proline carrier gene
  (repression by proline, catabolite repression, nitrogen control)
  　　Nakao et al., Mol. Gen. Genet. 214, 379-388 (1988)
• Purification and reconstitution of the proline carrier
  　　Hanada et al., J. Biol. Chem. 263, 7181-7185 (1988)
• Ordered binding model as transport reaction mechanism of secondary active transport systems
  　　Yamato and Rosenbusch, FEBS Lett. 151, 102-104 (1983)
  　　Fujimura et al., Biochemistry 22, 1954-1959 (1983)
  　　Yamato and Anraku, J. Membrane Biol. 114, 143-151 (1990)
• Structure and function of the proline carrier
  　　Yamato et al., J. Biol. Chem. 265, 2450-2455 (1990)

What we are going to do

• Structure and function of the proline carrier
  
• Effect of membrane potential on the transport reaction
  
• Development of utilization technique of the carrier proteins

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