Two highly conserved glutamate residues critical for type III sodium-dependent phosphate transport revealed by uncoupling transport function from retroviral receptor function

Type III sodium-dependent phosphate (NaPi) cotransporters, Pit1 and Pit2, have been assigned housekeeping Pi transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaPi cotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu(55) and Glu(575), with glutamine or with lysine severely impaired or knocked out, respectively, P-i transport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaPi cotransporters, in fungal NaPi cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaPi cotransport. Their putative transmembranic positions together with the presented data are consistent with Glu(55) and Glu(575) being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 P-i transport function.