Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli.
To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.
Opekarová
M
M
Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic. opekarov@biomed.cas.cz
Robl
I
I
Grassl
R
R
Tanner
W
W
D001692:Biological Transport; D002463:Cell Membrane Permeability; D002708:Chlorella / Q000235:genetics; D004875:Ergosterol / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells; D005947:Glucose / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011387:Prokaryotic Cells; D011510:Proteolipids; D011994:Recombinant Proteins / Q000378:metabolism; D027981:Symporters
D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; D027981:Symporters; C089820:monosaccharide-hydrogen cotransporter; C020485:proteoliposomes; D005947:Glucose; D004875:Ergosterol