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 of mediating inward Na⁺ currents in Xenopus laevis oocytes and K⁺ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K⁺ transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K⁺ channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na⁺ currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. 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 together, our results show that AtHKT1 contains eight transmembrane-spanning segments.