Omain, we generated three constructs expressing truncated loop7 domain determined by bioinformatics analysis (Supplementary Fig. S6a)28. Yeast two-hybrid analysis revealed that deletion of either Metyrosine Purity & Documentation residues 35682 or 23502 prevented binding with LC1, whilst mutants with deletion of residues 23555 and 40382 retained interaction with LC1 (Fig. 2e, Supplementary Fig. S6b). As a result, we constructed a series of mutants with C-terminal of loop7 truncated and located that only mutants containing residues 23502 could interact with LC1 (Fig. 2d, Supplementary Fig. S6c). To additional localize the binding website of LC1 in PiT2, we generated six overlapping 4-residue alanine substitution mutations within the loop7 domain involving residues 384 and 397. Among them, two alanine substitution mutants (residues 38689 A and 38890 A) failed to interact with LC1 (Fig. 2f, Supplementary Fig. S6c), which indicate that residues 38690 (YTCYT) are required for interaction amongst PiT2 and MAP1B. To further verify the interaction websites, we performed a co-immunoprecipitation experiment in Neuro2A cells. HA epitope tagged PiT28690A mutants were expressed in Neuro2A cells. Western blotting showed that LC1 antibody was able to pull down WT protein, but not 38690A or loop7 mutant proteins (Fig. 3f and Supplementary Fig. S3f). Prior analysis benefits have demonstrated that LC1 is linked for the membrane localization of some ion channels and transmembrane receptor292. Even so, by immunofluorescence assay, we located that WT and 386390A mutant PiT2 proteins localized to plasma membranes, and there have been no considerable differences in the fluorescence intensity in between WT and 38690 A mutant (Supplementary Fig. S1a,b). Mutating the MAP1B binding site in PiT2 impacts neurite length. Subsequent, we carried out immunofluorescence assay and discovered that overexpressing PiT2 (38690A) significantly decreased the length of neurites in Neuro2A cells (Fig. 4c,g). This indicates that mutating the MAP1B binding sites in loop7 results in reduced neurite length. We also evaluated whether transport function of PiT2 underlies the regulation of neurite outgrowth, and performed neuritogenesis assays in Neuro2A cells transfected with three PiT2 mutants discovered in IBGC families (S601W, R254and V507Efs2)13,33,34. The expression levels of 38691 A, S601W, R254and V507Efs2 mutant in Neuro2 cells were related (Supplementary Fig. S5b-c). Following induction of differentiation, Pi transport function deficient mutant PiT2-S601W (a missense mutation in C-PD domain) and PiT2-V507Efs2 (deleted C-PD domain only) didn’t impact the neurite outgrowth in Neuro2A cells (Fig. 4d,f,g). The mutant PiT2-R254 (deleted loop7 and C-PD domain) as comparable to PiT28690A and PiT2-loop7, major to significantly decreased length of neurites in Neuro2A cells (Fig. 4e,g). These final results demonstrate that loop7 domain of PiT2 might take part in the regulation of neurite outgrowth in Neuro2A cells, while PiT2 lacking Pi transport function exerts no impact on neurite outgrowth.The dPiT loop7 domain plays a Sulfentrazone Formula crucial role in dPiT function in vivo.Given that loop7 domain participates in PiT2 trafficking and regulates neurite outgrowth in Neuro2A cells, we examined if comparable functions exist in Drosophila. Sequence comparison showed that the Drosophila genome contains the homolog of human SLC20A2, CG42575, which encodes dPiT protein. Drosophila dPiT is 38 identical and 56 equivalent to human PiT2 (Supplementary Fig. S7b). We constructed dPiT loss of function mutants.