Li Xinjing, Zheng Jing, Su Jing, Wang Lin, Luan Lin, Wang Taotao, Bai Fang, Zhong Qing, Gong Qingqiu
State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China.
Department of Plant Biology and Ecology, College of Life Sciences, Nankai University, Tianjin, P. R. China.
Autophagy. 2025 Jan;21(1):141-159. doi: 10.1080/15548627.2024.2394302. Epub 2024 Sep 8.
Starvation- or stress-induced phosphatidylinositol 3-phosphate (PtdIns3P/PI3P) production at the endoplasmic reticulum (ER) subdomains organizes phagophore assembly and autophagosome formation. Coat protein complex II (COPII) vesicles budding from ER exit site (ERES) also contribute to autophagosome formation. Whether any PtdIns3P phosphatase functions at ERES to inhibit macroautophagy/autophagy is unknown. Here we report Myotubularin 2 (MTM2) of Arabidopsis as a PtdIns3P phosphatase that localizes to ERES and negatively regulates autophagy. MTM2 binds PtdIns3P with its PH-GRAM domain and acts toward PtdIns3P . Transiently expressed MTM2 colocalizes with ATG14b, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex, and overexpression of MTM2 blocks autophagic flux and causes over-accumulation of ATG18a, ATG5, and ATG8a. The mutant has higher levels of autophagy and is more tolerant to starvation, whereas overexpression leads to reduced autophagy and sensitivity to starvation. The phenotypes of are suppressed by mutation, suggesting that MTM2 acts upstream of ATG2. Importantly, MTM2 does not affect the endosomal functions of PtdIns3P. Instead, MTM2 specifically colocalizes with COPII coat proteins and is cradled by the ERES-defining protein SEC16. MTM2 interacts with SEC23A with its phosphatase domain and inhibits COPII-mediated protein secretion. Finally, a role for MTM2 in salt stress response is uncovered. resembles the halophyte in its efficient vacuolar compartmentation of Na, maintenance of chloroplast integrity, and timely regulation of autophagy-related genes. Our findings reveal a balance between PtdIns3P synthesis and turnover in autophagosome formation, and provide a new link between autophagy and COPII function.: ATG: autophagy related; BFA: brefeldin A; BiFC: bimolecular fluorescence complementation; CHX: cycloheximide; ConA: concanamycin A; COPII: coat protein complex II; ER: endoplasmic reticulum; ERES: ER exit site; MS: Murashige and Skoog; MTM: myotubularin; MVB: multivesicular body; PAS: phagophore assembly site; PI: phosphoinositide; TEM: transmission electron microscopy; WT: wild-type.
内质网（ER）亚结构域处由饥饿或应激诱导产生的磷脂酰肌醇3-磷酸（PtdIns3P/PI3P）可组织吞噬泡组装和自噬体形成。从内质网出口位点（ERES）出芽的II型被膜小泡蛋白复合物（COPII）也有助于自噬体形成。是否有任何PtdIns3P磷酸酶在ERES发挥作用以抑制巨自噬/自噬尚不清楚。在此，我们报道拟南芥的肌管蛋白2（MTM2）作为一种定位于ERES并负向调节自噬的PtdIns3P磷酸酶。MTM2通过其PH-GRAM结构域结合PtdIns3P并作用于PtdIns3P。瞬时表达的MTM2与磷脂酰肌醇3-激酶（PtdIns3K）复合物的一个亚基ATG14b共定位，MTM2的过表达会阻断自噬流并导致ATG18a、ATG5和ATG8a过度积累。MTM2突变体具有更高水平的自噬且对饥饿更耐受，而MTM2过表达则导致自噬减少和对饥饿敏感。MTM2突变体的表型被ATG2突变所抑制，表明MTM2在ATG2上游起作用。重要的是，MTM2不影响PtdIns3P的内体功能。相反，MTM2特异性地与COPII被膜蛋白共定位，并被定义ERES的蛋白SEC16所包裹。MTM2通过其磷酸酶结构域与SEC23A相互作用并抑制COPII介导的蛋白质分泌。最后，发现了MTM2在盐胁迫应答中的作用。MTM2突变体在Na的有效液泡区室化、叶绿体完整性维持以及自噬相关基因的及时调控方面类似于盐生植物。我们的研究结果揭示了自噬体形成过程中PtdIns3P合成与周转之间的平衡，并提供了自噬与COPII功能之间的新联系。：ATG：自噬相关；BFA：布雷菲德菌素A；BiFC：双分子荧光互补；CHX：环己酰亚胺；ConA： concanamycin A；COPII：II型被膜小泡蛋白复合物；ER：内质网；ERES：内质网出口位点；MS：Murashige和Skoog；MTM：肌管蛋白；MVB：多泡体；PAS：吞噬泡组装位点；PI：磷酸肌醇；TEM：透射电子显微镜；WT：野生型
