Putative topological model of human PiT2 and mutants. Putative membrane topology model of human PiT2 on which the mutant proteins investigated in the present paper are based; the model was originally proposed by O'Hara and coworkers [8, 20]. The numbers of the TMs are indicated above the model. Other membrane topology models have been proposed for PiT1 [22, 23] and PiT2 , which suggested diverging topology for the two paralogs; the alternative PiT2 model is shown in Figure 2. The amino acids previously identified in human PiT2 as being critical for Pi transport function are highlighted with black filling and pointed out with arrows; D28, E55, S113, D506, E575, and S593[18, 27, 28]. In human PiT1, the amino acids S128 (PiT2 S113) and S621 (PiT2 S593) have previously been identified as being critical for PiT1 Pi transport function . In the present study, human PiT2 H502 (situated in the PiT family signature sequence) and human PiT1 E70 (equivalent in position to human PiT2 E55) are also identified as critical for Pi transport function (see Figure 3). The grey-filled sequences (L11-L161 and V492-V640), represent the N- and C-terminal, respectively, ProDom domains (PD001131) published in 2004 defining the PiT family members . The dark grey-filed sequence (I53-L127) represents the most recent ProDom domain defining the PiT family members http://prodom.prabi.fr/.