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

中文翻译：

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AP-3囊泡脱膜发生在依赖HOPS的液泡束缚之后。

异四聚体衔接子（AP）复合物与小GTPase Arf1或脂质在货物选择，囊泡形成和真核细胞内膜出芽中协同作用。尽管大多数AP复合物还需要网格蛋白作为囊泡的外壳，但推测参与酵母中高尔基体到真空转运的AP-3涂层囊泡的形成依赖于液泡HOPS束缚复合物的亚基Vps41。HOPS也被认为是液泡中AP-3囊泡的系链。为了解开双重Vps41功能的难题，我们将Vps41稳定地锚定于线粒体外膜。通过监视AP-3募集，我们现在显示Vps41可以将AP-3囊泡束缚于线粒体，而在没有Vps41或网格蛋白的情况下仍可以形成AP-3囊泡。通过邻近标记和质谱，我们在AP-3涂层上鉴定了Arf1 GTPase激活蛋白（GAP）Age2，并表明在没有完全脱涂层的情况下可以发生束缚，但在液泡中不能融合。我们得出的结论是，AP-3囊泡出芽后仍保留其被膜，仅在空泡处束缚后才发生完全脱膜。