Abstract
The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increased Km value for D-glucose were isolated. The 40-fold increase in Km of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in Km for D-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the Km shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway. Whereas for most mutants obtained so far the Km change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the Km for D-glucose. Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group. Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential D-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose. The reverse substitution (I47N) decreases the affinity of HUP2 for D-galactose 20-fold.
Highlights
Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein
The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose
Support for this topological model comes from alkaline phosphatase fusion protein analysis of the Escherichia coli lactose permease lacY [11] and N-glycosylation scanning mutagenesis studies on the human glucose facilitator GLUT1 [12]
Summary
Strains and Growth Conditions—All cloning steps were carried out in E. coli DH5␣ with the plasmid vector pUC18. Transformation of S. pombe YGS-B25—Wild-type, point-mutated, and chimeric transporter cDNAs were cloned via SacI/BamHI into the shuttle vector pEVP11 [19] or pART3 [20], the latter allowing significant higher expression. The mutated HUP1 cDNAs were amplified afterward using flanking primers that bind in the promotor and the polylinker region of pEVP11 They were subcloned via SacI/ BamHI into pUC18 and their nucleotide changes were determined by sequence analysis using the T7SequencingTM kit (Pharmacia Biotech) and synthetic oligonucleotides. The SacI/KpnI fragment and the KpnI/BamHI fragment coding for the N- and C-terminal part were ligated to the respective missing sequences from the wild-type clone This resulted in HUP1 coding regions carrying either the one or the other mutation. Expression of mutant cDNAs was compared with that of wild-type HUP1 cloned in the same vector (pEVP11 or pART3)
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