Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.
The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.
Barrett
M P
MP
Université de Bordeaux II, France.
Tetaud
E
E
Seyfang
A
A
Bringaud
F
F
Baltz
T
T
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D006224:Cricetinae; D003847:Deoxyglucose / Q000378:metabolism; D005260:Female; D005632:Fructose / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011190:Potassium Cyanide / Q000494:pharmacology; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D014982:Xenopus laevis
D009004:Monosaccharide Transport Proteins; D015800:Protozoan Proteins; C079375:THT2 protein, Trypanosoma brucei; D005632:Fructose; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D003847:Deoxyglucose; D005947:Glucose; D011190:Potassium Cyanide