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Related Experiment Videos

Structure/function relationship of the Chlorella glucose/H+ symporter

T Caspari1, R Stadler, N Sauer

  • 1Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Germany.

The Journal of Biological Chemistry
|February 4, 1994
PubMed
Summary

The study engineered a glucose transporter (HUP1) in yeast, revealing that specific aspartic acid and glutamine residues are critical for its function. Mutations altering these key amino acids significantly impacted glucose transport activity and affinity.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Yeast Genetics

Background:

  • Hexose/H+ symporters are crucial for cellular glucose uptake.
  • Understanding the structure-function relationship of these transporters is vital for metabolic engineering and drug development.
  • The Chlorella kessleri HUP1 gene encodes a well-characterized hexose transporter.

Purpose of the Study:

  • To investigate the functional roles of specific amino acid residues in the Chlorella kessleri HUP1 hexose/H+ symporter.
  • To elucidate the structure-function relationships of key residues within transmembrane helices and conserved regions.
  • To characterize the impact of site-directed mutagenesis on transporter activity and substrate affinity.

Main Methods:

  • Expression of the Chlorella kessleri HUP1 gene in a glucose uptake-deficient Schizosaccharomyces pombe mutant.

Related Experiment Videos

  • Site-directed mutagenesis of conserved histidyl, aspartyl, and glutaminyl residues within the HUP1 transporter.
  • Assays for growth on glucose, 3-O-methylglucose accumulation, and kinetic analysis (Km values) of mutated transporters.

    • Main Results:

    • Mutations in histidyl residues (H73R, H170R, H495R) showed no significant effect on transport activity.
    • Replacement of Asp-44 with Asn or Glu drastically reduced or abolished transporter activity, respectively, and increased Km.
    • Mutations in conserved glutamine residues (Q179N, Q299N) significantly increased the Km value, indicating reduced substrate affinity.

    Conclusions:

    • Specific aspartic acid and glutamine residues are essential for the catalytic activity and substrate binding of the HUP1 transporter.
    • The study provides critical insights into the molecular mechanisms underlying hexose transport.
    • The developed yeast expression system serves as a valuable tool for functional analysis of transporter mutants.