Fig. 5.

ATP9A is required for Wls sorting and is required for Wnt gradient formation. a RPE-1 cells, stably expressing HA-WLS, under RNAi-mediated suppression of VPS35 or ATP9A were treated with Bafilomycin A1 (100 nM for 16 h). Knock-down of ATP9A was confirmed through RT-qPCR. b Quantification of HA-WLS protein levels reveal a similar loss of HA-WLS through VPS35 or ATP9A suppression that is reverted upon blocking of lysosomal-mediated degradation using Bafilomycin A1. Data presented as mean +/− s.e.m. from five independent biological replicates. Significance was determined using a one-way ANOVA followed by a post-hoc Dunnett’s test; *p < 0.05. c Tissue specific RNAi of tat-5 in Wnt producing cells (Pmig-14::tat-5 RNAi) in a vps-29(tm1320) sensitised mutant background. The percentage of animals with anteriorly displaced QL.d is shown (data are presented as mean +/− SD and include results from seven independent biological replicates, n ≥ 24 per replicate) *p = 0.013 (Student’s t-test). d Sequence alignment of Drs2p with Neo1p homologues highlighting the conserved catalytic glutamic acid residue essential for ATPase activity. e Overexpression of catalytically inactive TAT-5(E246Q) in a vps-29(tm1320)sensitised mutant background. The percentage of animals with anteriorly displaced QL.d is shown (data are presented as mean +/− SD and include results from three independent biological replicates, n ≥ 30 per replicate) *p = 0.0089 (Student’s t-test). f Schematic representation of our working model: SNX3-retromer, coupled to MON2/DOPEY2, enriches Wntless into nascent carriers, formed through the flippase activity of ATP9A