The P4‐ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4‐ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4‐ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole‐specific subunit of TRAPPIII, to the N‐terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4‐ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4‐ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4‐ATPases in MTC regulation. Synopsis Binding of Trs85 to the I(S/R)TTK motif in the N‐terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9. This is required for Atg9 delivery to the pre‐autophagosomal structure along the Cytoplasm‐to‐vacuole targeting pathway. P4‐ATPases, a family of lipid flippases, interact with Multisubunit Tethering Complexes (MTCs) in yeast. Binding of MTC subunits to P4‐ATPases requires the I(S/R)TTK motif of the flippases. The P4‐ATPase Drs2 stabilizes TRAPPIII on Atg9‐loaded compartments to facilitate transport of Atg9 vesicles. The regulatory role of Drs2 is critical for the Cytoplasm‐to‐vacuole targeting pathway at low temperatures. Graphical Abstract Binding of Trs85 to the I(S/R)TTK motif in the N‐terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9. This is required for Atg9 delivery to the pre‐autophagosomal structure along the Cytoplasm‐to‐vacuole targeting pathway.