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. OG was funded by research grants from the Spanish funding agency (PGC2018-095745-B-I00, EUR2019-103815 and PID2021-127773NB-I00/FEDER UE and supported by the “Unidad de Excelencia María de Maeztu” (ref: MDM-2014-0370) funded by the MCIN/AEI/10.13039/501100011033) and a Research Grant from HFSP (ref: RGP0017/2020). The proteomics analyses were performed in the CRG/UPF Proteomics Unit which is part of the of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013-2016, funded by ISCIII and ERDF. We thank Josep Vilardell, Bernat Crosas, Amy Curwin, Vivek Malhotra, Michael Knop, Marko Kaksonen, Kenji Maeda, Manuel García-Muñiz and Charles Boone for providing devices, reagents, strains and plasmids. We thank the Advanced Digital Microscopy Core Facility (ADMCF), the Biostatistics/Bioinformatics and the Mass Spectrometry and Proteomics facilities of the IRB Barcelona for live-cell imaging, technical support and also sharing devices with us. We thank Simona Barankova, Radovan Dojcilovic and Daniel Castaño-Díez for fruitful discussions. We thank Raúl Méndez and Ignasi Fita for their support and mentoring during these years. IP was supported by FPI awarded by the Spanish Ministry of Economy and Competitiveness (MINECO) (ref: BES-2013-063945) and EMBO Short-Term Fellowship 7076.
