ACTIVE SUGAR-TRANSPORT IN EUKARYOTES

Sugar transporters in prokaryotes and eukaryotes belong to a large fam ily of membrane proteins containing 12 transmembrane alpha-helices. Th ey are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are g enerated and maintained by ion pumps. The facilitative and H+-driven s ugar transporters belong to a gene family with a distinctive secondary structure profile. The Na+-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na+- and H+-driven sugar cotr ansporters share common transport mechanisms. To explore these mechani sms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT ) in Xenopus laevis oocytes and measured the kinetics of sugar transpo rt using two-electrode voltage-clamp techniques. For SGLT1, we have de veloped a six-state ordered model that accounts for the experimental d ata. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rat e constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural stud ies have confirmed these estimates of SGLT1 density. (ii) We have pert urbed the kinetics of the cotransporter by site-directed mutagenesis o f selected residues. For example, we have identified a charged residue which dramatically changes the kinetics of charge transfer. (iii) We have examined the kinetics of sugar and Na+ analogs. The V-max of suga r transport decreases dramatically with bulky phenyl glucosides and in creases when H+ replaces Na+. These results permit us to extend and re fine our model for transport. The model has been useful in the analysi s of mutant SGLT1 proteins: in the case of a D176A mutant, the primary effect is to alter rates of conformational changes of the unloaded pr otein, and with the glucose/galactose malabsorption syndrome mutant D2 8N SGLT1, the mutation disrupts the delivery of SGLT1 glycosylated pro tein from the endoplasmic reticulum to the plasma membrane.