The P4-ATPase Drs2 regulates homeostasis of Atg9

Atg9 is a transmembrane protein essential for selective autophagy, a pathway that mediates the targeted degradation of cellular components to sustain the cell fitness. To preserve the functionality of this pathway, the cell adjusts the transport of vesicles loaded with Atg9 through mechanisms that are not understood. Here we used live-cell imaging to investigate the interactome that regulates Multisubunit Tethering Complexes (MTCs), a set of conserved protein complexes that control vesicle tethering. We found that P4-ATPases, a family of lipid transporters involved in the biogenesis of vesicles, interact with MTCs that participate in the transport of Atg9, such as TRAPPIII. Using the lipid flippase Drs2, we demonstrated that the I(S/R)TTK motif nested in the N-terminal tail cavity of P4-ATPases is necessary for the interaction with MTCs and to maintain the homeostasis of Atg9. At low temperature, the cell enhances the assembly of the Drs2-TRAPPIII module and Drs2 is fundamental for the early stages of selective autophagy, a function that is independent from its activity as lipid flippase and its role in other vesicle transport pathways. Selective autophagy is a catabolic process that targets the degradation of cellular components, such as organelles, protein aggregates or pathogens, in a specific manner. Selective autophagy requires the de novo formation of double membrane compartments, generally called autophagosomes, to confine the selected cargo and to deliver it to the vacuole/lysosomes for subsequent processing. Selective autophagy has a main role in preserving the cell fitness in front of pathogens or stress, but it is also necessary to maintain the cell biology in processes such as endocytosis. The Autophagy-related protein 9 (Atg9) is a transmembrane protein that has recently been characterized as a lipid scramblase , and it is essential for the formation of the double membrane engulfing the cargo. In growing cells, Atg9 localizes at endocytic compartments, the Golgi and numerous cytoplasmic vesicular structures, which move quickly throughout the cytoplasm. When required, a fraction of Atg9 is mobilized to the pre-autophagosomal structure (PAS), where autophagosome biogenesis takes place. Despite the main role of Atg9, the mechanisms regulating Atg9 homeostasis and its trafficking in the endomembrane system remain unclear. Multisubunit Tethering Complexes (MTCs) are a group of protein complexes essential for vesicle transport, including the trafficking of Atg9 vesicles. MTCs mediate the tethering of the vesicle to the acceptor membrane through long-range interactions that precede vesicle fusion and cargo delivery at the destination of the transport pathway. Eight MTCs have been described in yeast, all conserved in humans. The conserved oligomeric Golgi (COG) complex is a heterooctamer involved in intraGolgi retrograde transport. Dsl1, with only three different subunits, is responsible for tethering COPI vesicles derived from the Golgi to the endoplasmic reticulum (ER). The class C core vacuole/endosome tethering (CORVET) complex and the homotypic fusion and vacuole protein sorting (HOPS) complex share a common core of four subunits and they have two additional specific subunits each. Both complexes act at the endosomal/vacuolar pathway where CORVET tethers vesicles at early endosomes and HOPS functions in late endosomes and the vacuole.