Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters

The Arabidopsis thaliana AtHKT1 protein, a Na+/K+ transporter, is capable o f mediating inward Na+ currents in Xenopus laevis oocytes and K+ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K+ transpor ters. These proteins have been proposed to contain eight transmembrane segm ents and four pore-forming regions arranged in a mode similar to that of a K+ channel tetramer. However, computer analysis of the AtHKT1 sequence iden tified eleven potential transmembrane segments. We have investigated the me mbrane topology of AtHKT1 with three different techniques. First, a gene fu sion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane fold ing of the C-terminal region had to be refined. Second, with a reticulocyte -lysate supplemented with dog-pancreas microsomes, we demonstrated that N-g lycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na+ currents in X. laevis oocytes with th e same characteristics as the wild-type protein, indicating that N-glycosyl ation is not essential for the functional expression and membrane targeting of AtHKT1, Five potential glycosylation sites were introduced into the N42 9Q. Their pattern of glycosylation supported the model based on the E. coli -alkaline phosphatase data. Third, immunocytochemical experiments with FLAG -tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135-142 and 377-384, face the cytosol, whereas the region of residues 55-62 is exposed to the outside. Taken toge ther, our results show that AtHKT1 contains eight transmembrane-spanning se gments.