Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, School of Basic Medical Sciences, Guangzhou Medical University, 511436, Guangzhou, China.
State Key Laboratory of Respiratory Disease, Guangzhou Medical University, 511436, Guangzhou, China.
Autophagy. 2021 May;17(5):1142-1156. doi: 10.1080/15548627.2020.1749490. Epub 2020 Apr 19.
Energy deprivation activates the cellular energy sensor AMP-activated protein kinase (AMPK), which in turn induces macroautophagy/autophagy. The mitochondrial-associated ER membrane (MAM) plays a key role in mitochondrial division and autophagy, and the mitochondrial fusion protein MFN2 (mitofusin 2) tethers the MAM, but the mechanism by which AMPK and MFN2 regulate autophagy in response to energy stress remains unclear. Here, we found that energy stress not only triggers mitochondrial fission and autophagy, but more importantly increases the number of MAMs, a process that requires AMPK. Interestingly, under energy stress, considerable amounts of AMPK translocate from cytosol to the MAM and the mitochondrion as mitochondrial fission occurs. Unexpectedly, AMPK interacts directly with MFN2. The autophagic ability of mouse embryonic fibroblasts (MEFs) lacking MFN2 () is significantly attenuated in response to energy stress as compared to wild-type MEFs (WT MEFs), while re-expression of MFN2 in cells rescues the autophagy defects of these cells. The abundance of MAMs is also greatly reduced in MFN2-deficient cells. Functional experiments show that the oxygen consumption rate and the glycolytic function of cells lacking MFN2 but not MFN1 are obviously attenuated, and MFN2 is important for cell survival under energy stress. In conclusion, our study establishes the molecular link between the energy sensor AMPK and the MAM tether MFN2, and reveals the important role of AMPK and MFN2 in energy stress-induced autophagy and MAM dynamics. ACTB, actin beta; AMPK, AMP-activated protein kinase; BECN1, beclin 1; CANX, calnexin; ER, endoplasmic reticulum; HRP, horseradish peroxidase; EM, electron microscopy; FL, full-length; KD, kinase dead, KO, knockout; MAb, monoclonal antibody; MAMs, mitochondria-associated membranes; MAP1LC3/LC3B, microtubule associated protein 1 light chain 3; MFN2, mitofusin 2; OPA1, OPA1 mitochondrial dynamin like GTPase; PAb, polyclonal antibody; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; SD, standard deviation; TEM, transmission electron microscopy; TOMM20, translocase of outer mitochondrial membrane 20; ULK1, unc-51 like autophagy activating kinase 1; MEF, mouse embryonic fibroblast; WT, wildtype.
能量剥夺激活细胞能量传感器 AMP 激活蛋白激酶（AMPK），进而诱导巨自噬/自噬。线粒体相关内质网膜（MAM）在线粒体分裂和自噬中起关键作用，线粒体融合蛋白 MFN2（线粒体融合蛋白 2）将 MAM 连接起来，但 AMPK 和 MFN2 调节自噬以响应能量应激的机制尚不清楚。在这里，我们发现能量应激不仅触发线粒体裂变和自噬，而且更重要的是增加 MAMs 的数量，这一过程需要 AMPK。有趣的是，在能量应激下，相当数量的 AMPK从细胞质转移到 MAM 和线粒体，随着线粒体裂变的发生。出乎意料的是，AMPK 与 MFN2 直接相互作用。与野生型 MEF（WT MEF）相比，缺乏 MFN2 的小鼠胚胎成纤维细胞（MEF）在能量应激下的自噬能力明显减弱，而在 细胞中重新表达 MFN2 则可以挽救这些细胞的自噬缺陷。MFN2 缺乏细胞中的 MAMs 丰度也大大降低。功能实验表明，缺乏 MFN2 但不缺乏 MFN1 的细胞的耗氧率和糖酵解功能明显减弱，MFN2 对于细胞在能量应激下的存活很重要。总之，我们的研究建立了能量传感器 AMPK 与 MAM 连接蛋白 MFN2 之间的分子联系，并揭示了 AMPK 和 MFN2 在能量应激诱导的自噬和 MAM 动力学中的重要作用。ACTB，肌动蛋白 β；AMPK，AMP 激活蛋白激酶；BECN1，自噬相关蛋白 12；CANX，钙网织蛋白；ER，内质网；HRP，辣根过氧化物酶；EM，电子显微镜；FL，全长；KD，激酶失活；KO，敲除；MAb，单克隆抗体；MAMs，线粒体相关膜；MAP1LC3/LC3B，微管相关蛋白 1 轻链 3；MFN2，线粒体融合蛋白 2；OPA1，OPA1 线粒体动力相关 GTP 酶；PAb，多克隆抗体；PtdIns3K，III 类磷酸肌醇 3-激酶；PtdIns3P，磷酸肌醇 3-磷酸；SD，标准偏差；TEM，透射电子显微镜；TOMM20，外线粒体膜转运蛋白 20；ULK1，UNC-51 样自噬激活激酶 1；MEF，小鼠胚胎成纤维细胞；WT，野生型。
