Autophagy is a complex intracellular degradation pathway that is highly conserved across eukaryotes. This pathway relies on the coordinated interplay between the core autophagy machinery and diverse membrane sources to mediate the de novo formation of double-membrane vesicles, known as autophagosomes, which sequester cytoplasmic material for subsequent degradation. Golgi-derived Atg9-containing vesicles are critical for autophagy, serving as membrane donors that initiate autophagosome biogenesis. In Saccharomyces cerevisiae, these vesicles incorporate the multi-pass transmembrane protein Atg9, the single-pass transmembrane protein Atg27, and the peripheral membrane protein Atg23. However, the organisation of these proteins within Atg9-containing vesicles, the functions of their direct protein-protein interactions, and the regulation of these interactions by the autophagy machinery remain unclear. To address these questions, we used a combination of in vitro reconstitution, structure prediction, mass spectrometry, biochemistry and cell biology approaches to systematically dissect the protein-protein interactions among Atg9, Atg23, and Atg27, and uncover their regulation in space and time. We find that Atg23 engages Atg9 using a bipartite binding mode, providing a structural model for how Atg23 promotes the budding of Atg9-containing vesicles. Our findings furthermore reveal that Atg1-dependent phosphorylation of Atg9 remodels its interactions with Atg23 and Atg27 at the phagophore assembly site, which is essential for autophagy initiation. Together, these findings establish a molecular and regulatory framework for the early stages of autophagosome biogenesis.