PDF(Pubmed)
Abstract：
The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion.Abbreviations: Cardiolipin: 1\',3\'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1\'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2: 1,2-dioleoyl-sn-glycero-3-phospho-(1\"-myo-inositol-3\',5\'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1\'-myo-inositol-3\'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1\"-myo-inositol-4\'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17.
摘要：
自噬小体SNARESTX17（Syntaxin17）促进溶酶体融合和降解，但它的自噬体募集尚不完全清楚。值得注意的是,PtdIns4P在自噬体上产生并通过未知机制促进融合。在这里，我们显示可溶性重组STX17自发募集到带负电荷的脂质体中，并且将PtdIns4P添加到含有中性脂质的脂质体中足以募集。始终如一，STX17在细胞中与PtdIns4P阳性自噬体共定位，和PtdIns4P在自噬体上的合成的特异性抑制阻止其负载。分子动力学模拟表明C-末端带正电荷的氨基酸与含有带负电荷的PtdIns4P的膜双层建立接触。因此，STX17C末端Lys和Arg残基的Ala取代消除了膜结合并损害了其自噬体募集。最后,只有野生型而不是Ala取代的STX17表达可以挽救STX17功能丧失细胞的自噬-溶酶体融合缺陷。因此，我们确定了促进溶酶体融合的自噬体成熟的关键步骤。
