The endosomal compartment is crucial for the molecular fine-tuning of Wnt signaling. This is not only true for the signal-receiving but also for the Wnt secreting cell. We have shown that a portion of Wnt3A is secreted on exosomes via multivesicular bodies (MVBs) and that its sorting depends on the unusual v-SNARE Ykt6, the only SNARE protein lacking a transmembrane domain. Ykt6 is involved in vesicle fusion events at the Golgi and MVBs, to where it cycles by an autocatalytic, reversible palmitoylation. We found that depletion of Ykt6 leads to loss of Wnt3A secretion and rerouting of exosomal cargo into lysosomes. In the proposed project we want to understand the trafficking dynamics of Wnt3A from the Golgi apparatus into MVBs using the Ykt6 phenotype and characterized cycling-defect Ykt6 mutants. We will use a cellular system for releasable protein secretion (RUSH), biochemical assays (secretion, surface biotinylation, Proteinase K protection) and confocal microscopy (live-imaging, colocalization studies and TIRF) to study the passage of Wnt3A and its cargo receptor Evi through the secretory pathway. Since Ykt6 is involved in Wnt sorting in the secreting cell but not in the receiving cell, we hypothesize that Wnt/Evi cargo complexes are recognized and sorted into MVBs by Ykt6-containing complexes. To analyze how Wnt3A-containing vesicles are recognized on the cytoplasmic face we will perform co-immunoprecipitation studies of cycling-defect Ykt6 mutants and Evi deletion constructs. New factors involved in the Ykt6-mediated sorting will be identified by a proteomics approach using Ykt6 co-immunoprecipitations and/or a proximity-dependent labeling assay. Further characterization of these factors in cell culture will enable us to investigate the Ykt6-mediated sorting mechanism of exosomal Wnt3A. Finally, the approach in human cells will be complemented with an in vivo study of Ykt6 in Drosophila melanogaster. We will characterize two Ykt6 mutant lines with clear Wnt signaling phenotypes for long-range effects of exosomal Wg and expand the functional analysis to evolutionary conserved interactors of Ykt6 identified in the second part. Our approach will elucidate the cell biological mechanism behind regulated Wnt secretion via endosomes.