ABSTRACT

Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. Here, we discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1⁺ vesicles during starvation-induced autophagy. We further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. Our findings highlight the important role of PTPN9 in the regulation of ATG16L1⁺ autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status.

Abbreviations: csw: corkscrew; EBSS: Earle’s balanced salt solution; ERGIC: ER-Golgi intermediate compartment; ESCRT: endosomal sorting complexes required for transport; mop: myopic; NSF: N-ethylmaleimide-sensitive factor; PAS: phagophore assembly site; PolyQ: polyglutamine; PtdIns3P: phosphatidylinositol-3-phosphate; PTK: protein tyrosine kinase; PTM: posttranslational modification; PTP: protein tyrosine phosphatase; PTPN23/HD-PTP: protein tyrosine phosphatase non-receptor type 23; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; STX7: syntaxin 7; STX8: syntaxin 8; STX17: syntaxin 17; VAMP3: vesicle associated membrane protein 3; VAMP7: vesicle associated membrane protein 7; VTI1B: vesicle transport through interaction with t-SNAREs 1B; YKT6: YKT6 v-SNARE homolog; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

摘要

巨自噬/自噬是一种进化上保守的细胞内降解细胞质材料的途径。在应激条件下，自噬上调，吞噬细胞扩张形成双膜自噬体。ATG16L1 前体融合有助于吞噬细胞结构的发育，对自噬体的生物发生至关重要。在这里，我们发现了蛋白酪氨酸磷酸酶 PTPN9 在调节同型 ATG16L1 囊泡融合和早期自噬体形成中的新作用。PTPN9 及其果蝇同源物 Ptpmeg2 的消耗损害了自噬体的形成和自噬通量。PTPN9 与 ATG16L1 共定位，对于 ATG16L1 ^{+的同型融合至关重要}饥饿诱导的自噬过程中的囊泡。我们进一步将 Q-SNARE VTI1B 鉴定为 PTPN9 磷酸酶的底物靶标。与 PTPN9 一样，VTI1B 非磷酸化突变体而非拟磷突变体增强了 SNARE 复合物组装和自噬通量。我们的研究结果强调了 PTPN9 通过调节 VTI1B 磷酸化状态在调节 ATG16L1 ⁺自噬体前体融合和自噬体生物发生中的重要作用。

缩写：csw：开瓶器；EBSS：厄尔平衡盐溶液；ERGIC：ER-高尔基中间隔室；ESCRT：运输所需的内体分选复合物；拖把：近视；NSF：N-乙基马来酰亚胺敏感因子；PAS：吞噬细胞组装位点；PolyQ：聚谷氨酰胺；PtdIns3P：3-磷酸磷脂酰肌醇；PTK：蛋白酪氨酸激酶；PTM：翻译后修饰；PTP：蛋白酪氨酸磷酸酶；PTPN23/HD-PTP：蛋白酪氨酸磷酸酶非受体 23 型；SNARE：可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体；STX7：语法 7；STX8：语法 8；STX17：语法 17；VAMP3：囊泡相关膜蛋白 3；VAMP7：囊泡相关膜蛋白 7；VTI1B：通过与 t-SNARE 1B 相互作用的囊泡运输；YKT6：YKT6 v-SNARE 同源物；ZFYVE1/DFCP1：锌指FYVE型含1个。