Structure/function relationship of the Chlorella glucose/H+ symporter.
The Clorella kessleri HUP 1 gene coding for a hexose/H+ symporter has been expressed in a glucose uptake-deficient mutant of Schizosaccharomyces pombe. The transformants are able to grow on glucose and to accumulate 3-O-methylglucose 100-fold. This system has been used to test the activity of specifically mutated HUP 1 cDNAs. All three histidyl residues were exchanged with arginine (H73R, H170R, and H495R) without a major effect on transport activity. When Asp-44 within the first transmembrane helix was replaced by Asn, the transporter was inactive; replacement by Glu (D44E) resulted in a loss of activity by 90% and a 15-fold increased Km value. Glutamine residues conserved in all glucose transporters sequenced so far were exchanged: Q179N (in helix 5), Q298G and Q299N (both in helix 7). Whereas Q298G only resulted in a small Km change, both Q179N and Q299N showed an increase in Km by a factor of 10. Inserting 4 additional amino acids each into the two largest loops (1 and 6) reduced the activity dramatically; only in the latter case this was due to decreased protein synthesis or stability. Two COOH-terminal deletions (-27 and -43 amino acids) were also tested. The 27 COOH-terminal amino acids, but not the 43 COOH-terminal amino acids, could be removed without affecting transporter activity.
Caspari
T
T
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Germany.
Stadler
R
R
Sauer
N
N
Tanner
W
W
D019325:3-O-Methylglucose; D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D015153:Blotting, Western; D002708:Chlorella / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019325:3-O-Methylglucose; D005947:Glucose