The autophagy-specific exocyst subcomplex contributes to phagophore assembly site integrity by promoting phagophore expansion.

Autophagy is a crucial intracellular pathway for maintaining cellular homeostasis. It involves forming double-membrane vesicles called autophagosomes, which deliver cytosolic cargoes to the lysosomes/vacuoles for degradation. Biogenesis of autophagosomes is a membrane-intensive process wherein the membrane expansion steps are poorly understood. The tethering complex, exocyst, canonically implicated in secretion, also participates in autophagosome biogenesis in yeast, plants, and mammals. However, the contribution of the exocyst complex in autophagosome biogenesis is unclear. In this study, using yeast temperature-sensitive mutants of the exocyst, we observed the accumulation of multiple abortive PAS incapable of autophagosome biogenesis. These dysfunctional abortive structures were enriched with core autophagy proteins involved in initiation and membrane expansion. However, the membrane expansion ability required for cargo capture was severely compromised in these mutants. Further investigations, including a comprehensive epistasis analysis, revealed that the exocyst plays a role downstream of the Atg1 complex. However, it was required at a stage before phosphatidylinositol 3-kinase (PI3K complex I), Atg2-Atg18, and Atg12-Atg5-Atg16 complexes. Taken together, we show that productive PAS formation and membrane expansion during autophagosome biogenesis are exquisitely orchestrated by the autophagy-specific exocyst subcomplex, which excludes Exo70.