Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether transported vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are just partially understood. Here we systematically investigate the interactome that regulates the function of MTCs. We found that P4-ATPases, a family of lipid transporters involved in the biogenesis of vesicles, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We used the lipid flippase Drs2 as a model to investigate the mechanism and biological relevance of such interplay during the transport of Atg9 vesicles. Binding to the N-terminal tail of Drs2 stabilizes TRAPPIII on membrane compartments loaded with Atg9 and it is required for the delivery of Atg9 during selective autophagy, a role that is independent of previously reported functions of the P4-ATPase. This mechanism relies on the I(S/R)TTK motif nested in the N-terminal tail cavity of Drs2, a motif that is required for the interaction with MTCs.
