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Vac8 determines phagophore assembly site vacuolar localization during nitrogen starvation-induced autophagy

ABSTRACT Macroautophagy/autophagy is a key catabolic process in which different cellular components are sequestered inside double-membrane vesicles called autophagosomes for subsequent degradation. In yeast, autophagosome formation occurs at the phagophore assembly site (PAS), a specific perivacuolar location that works as an organizing center for the recruitment of different autophagy-related (Atg) proteins. How the PAS is localized to the vacuolar periphery is not well understood. Here we show that the vacuolar membrane protein Vac8 is required for correct vacuolar localization of the PAS. We provide evidence that Vac8 anchors the PAS to the vacuolar membrane by binding Atg13 and recruiting the Atg1 initiation complex. VAC8 deletion or mislocalization of the protein reduce autophagy activity, highlighting the importance of both the PAS and the correct vacuolar localization of the Atg1 initiation complex for efficient and robust autophagy. Abbreviations: AID: auxin-inducible degradation; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; GFP: green fluorescent protein; IAA: 3-indole acetic acid; PAS: phagophore assembly site; RFP: red fluorescent protein