Transmembrane topology of PiT-2, a phosphate transporter-retrovirus receptor

PiT-1 and PiT-2 are related multiple transmembrane proteins which function as sodium-dependent phosphate transporters and as the cell receptors of sev eral oncoretroviruses, Two copies of a homology domain that is found in dis tantly related species assign these proteins to a large family of phosphate transporters. A current membrane topology model of PiT-1 and PiT-2 predict s 10 transmembrane domains. However, the validity of this model has not bee n addressed experimentally. We addressed this issue by a comprehensive stud y of human PiT-2, Evidence was obtained for glycosylation of asparagine 81. Epitope tagging showed that the N- and C-terminal extremities are extracel lular, The orientation of C-terminal-truncation mutants expressed in cell-f ree translation assays and incorporated into microsomal membranes was exami ned by immunoprecipitation, Data were interpreted with respect to previous knowledge about retrovirus binding sites, to the existence of repeated homo logy domains, and to predictions made in family members. A model in which P iT-2 has 12 transmembrane domains and extracellular N- and C-terminal extre mities is proposed, This model, which differs significantly from previous p redictions about PiT-2 topology, may be useful for further investigations o f PiT-2 interactions with other proteins and for the understanding of PiT-2 transporter and virus receptor functions.