AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering

Abstract

Heterotetrameric adapter (AP) complexes cooperate with the smallGTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While mostAPcomplexes also require clathrin as the outer vesicle shell, formation ofAP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolarHOPStethering complex.HOPShas also been identified as the tether ofAP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoringAP-3 recruitment, we now show that Vps41 can tetherAP-3 vesicles to mitochondria, yetAP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1GTPase-activating protein (GAP) Age2 at theAP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude thatAP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole.