Department of Biology/Chemistry, Biochemistry Section, University of Osnabrück, Osnabrück, Germany.
Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
EMBO J. 2020 Oct 15;39(20):e105117. doi: 10.15252/embj.2020105117. Epub 2020 Aug 25.
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.
异源四聚体衔接（AP）复合物与小 GTPase Arf1 或脂质在真核细胞的内膜中协同作用，参与货物选择、囊泡形成和出芽。虽然大多数 AP 复合物也需要网格蛋白作为外囊泡壳，但酵母中高尔基体到液泡运输所涉及的 AP-3 包被囊泡的形成被假定依赖于液泡相关 HOPS 连接复合物的 Vps41 亚基。HOPS 也被确定为 AP-3 囊泡在液泡上的连接物。为了解决 Vps41 双重功能的难题，我们将 Vps41 稳定地锚定在线粒体的外膜上。通过监测 AP-3 的募集，我们现在表明 Vps41 可以将 AP-3 囊泡连接到线粒体上，然而，在没有 Vps41 或网格蛋白的情况下也可以形成 AP-3 囊泡。通过邻近标记和质谱分析，我们在 AP-3 外壳上鉴定出 Arf1 GTP 酶激活蛋白（GAP）Age2，并表明在没有完全脱壳的情况下可以发生连接，但不能发生融合。我们得出结论，AP-3 囊泡在出芽后保留其外壳，并且只有在与液泡连接后才会完全脱壳。
