The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore

MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12–ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation. <b>Abbreviations:</b> CQ: Chloroquine; EBSS: Earle’s Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1.

MOAP1（modulator of apoptosis 1，细胞凋亡调节因子1）是一种与BAX结合的蛋白质，受泛素-蛋白酶体系统（ubiquitin-proteasome system）的严格调控。凋亡刺激可稳定MOAP1蛋白，并促进其与BAX的相互作用，进而介导细胞凋亡的发生。在此我们发现，与对凋亡刺激产生抗性的情况相反，MOAP1基因敲除细胞对EBSS（Earle’s平衡盐溶液，Earle’s Balanced Salt Solution）处理引发的饥饿诱导性细胞死亡表现出超敏反应。MOAP1基因敲除细胞在EBSS诱导的巨自噬（macroautophagy，又称自噬）信号通路中存在功能缺陷。机制分析结果显示，在EBSS处理后，MOAP1基因敲除细胞在自噬前体相关的磷脂酰肌醇-3-磷酸（phosphatidylinositol-3-phosphate，PtdIns3P）结合蛋白ZFYVE1/DFCP1和WIPI2的招募过程中，以及在由ATG12–ATG5-ATG16L1复合体调控的LC3脂化机制中，均未出现明显缺陷。有趣的是，在经EBSS处理的细胞中，MOAP1对于促进自噬泡（phagophore）的高效闭合至关重要。通过Halo标记LC3的自噬体完成实验对LC3阳性膜结构进行分析后发现，经EBSS处理的MOAP1基因敲除细胞中，主要存在的是未闭合的自噬泡，而非已闭合的自噬体。通常在EBSS处理条件下会被包裹在闭合自噬体内的自噬底物SQSTM1/p62，在MOAP1缺失的情况下，也极易被蛋白酶K（proteinase K）降解。MOAP1可与LC3结合，且该结合过程严格依赖于其N端区域存在的LC3相互作用区域（LC3-interacting region，LIR）基序。在MOAP1基因敲除细胞中重新表达野生型MOAP1，而非其LC3结合缺陷突变体MOAP1-LIR，可恢复EBSS诱导的自噬过程。综上，上述实验结果表明，MOAP1在饥饿状态下通过与LC3结合促进自噬泡的高效闭合，在自噬过程中发挥独特的调控作用。

**缩写说明：** CQ：氯喹（Chloroquine）；EBSS：Earle’s平衡盐溶液（Earle’s Balanced Salt Solution）；GABARAP：γ-氨基丁酸受体相关蛋白（Gamma-Amino Butyric Acid Receptor Associated Protein）；IF：免疫荧光（Immunofluorescence）；IP：免疫沉淀（Immunoprecipitation）；LAMP1：溶酶体相关膜蛋白1（Lysosomal-Associated Membrane Protein 1）；LIR：LC3相互作用区域（LC3-Interacting Region）；MAP1LC3/LC3：微管相关蛋白1轻链3（Microtubule Associated Protein 1 Light Chain 3）；MEF：小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblast）；MOAP1：细胞凋亡调节因子1（Modulator of Apoptosis 1）；PE：磷脂酰乙醇胺（Phosphatidylethanolamine）；PtdIns3K：III类PtdIns3K复合体I（class III PtdIns3K complex I）；PtdIns3P：磷脂酰肌醇-3-磷酸（Phosphatidylinositol-3-phosphate）；STX17：突触融合蛋白17（Syntaxin 17）；ULK1：unc-51样自噬激活激酶1（unc-51 like autophagy activating kinase 1）。