Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Department of Molecular and Cell Biology and California Institute of Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720.
Mol Biol Cell. 2020 Apr 15;31(9):963-979. doi: 10.1091/mbc.E19-11-0658. Epub 2020 Feb 19.
The heterotetrameric adaptor protein complex 4 (AP-4) is a component of a protein coat associated with the -Golgi network (TGN). Mutations in AP-4 subunits cause a complicated form of autosomal-recessive hereditary spastic paraplegia termed AP-4-deficiency syndrome. Recent studies showed that AP-4 mediates export of the transmembrane autophagy protein ATG9A from the TGN to preautophagosomal structures. To identify additional proteins that cooperate with AP-4 in ATG9A trafficking, we performed affinity purification-mass spectrometry followed by validation of the hits by biochemical and functional analyses. This approach resulted in the identification of the fused toes homolog-Hook-FHIP (FHF) complex as a novel AP-4 accessory factor. We found that the AP-4-FHF interaction is mediated by direct binding of the AP-4 μ4 subunit to coiled-coil domains in the Hook1 and Hook2 subunits of FHF. Knockdown of FHF subunits resulted in dispersal of AP-4 and ATG9A from the perinuclear region of the cell, consistent with the previously demonstrated role of the FHF complex in coupling organelles to the microtubule (MT) retrograde motor dynein-dynactin. These findings thus uncover an additional mechanism for the distribution of ATG9A within cells and provide further evidence for a role of protein coats in coupling transport vesicles to MT motors.
四聚体衔接蛋白复合物 4（AP-4）是与高尔基体网络（TGN）相关的蛋白被膜的组成部分。AP-4 亚基的突变会导致一种称为 AP-4 缺乏综合征的常染色体隐性遗传性痉挛性截瘫的复杂形式。最近的研究表明，AP-4 介导跨膜自噬蛋白 ATG9A 从 TGN 向前自噬体结构的输出。为了鉴定与 AP-4 在 ATG9A 运输中合作的其他蛋白质，我们进行了亲和纯化-质谱分析，随后通过生化和功能分析对命中物进行了验证。这种方法导致鉴定出融合脚趾同源物-Hook-FHIP（FHF）复合物作为一种新的 AP-4 辅助因子。我们发现，AP-4-FHF 相互作用是由 AP-4 μ4 亚基与 FHF 的 Hook1 和 Hook2 亚基中的卷曲螺旋结构域的直接结合介导的。FHF 亚基的敲低导致 AP-4 和 ATG9A 从细胞的核周区域分散，这与 FHF 复合物在将细胞器与微管（MT）逆行马达动力蛋白 dynein-dynactin 偶联的先前证明的作用一致。这些发现因此揭示了细胞内 ATG9A 分布的另一种机制，并为蛋白被膜在将运输小泡与 MT 马达偶联中的作用提供了进一步的证据。
