College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei, China.
Autophagy. 2012 Feb 1;8(2):213-21. doi: 10.4161/auto.8.2.18563.
Supplementation of branched chain amino acids, especially leucine, is critical to improve malnutrition by regulating protein synthesis and degradation. Emerging evidence has linked leucine deprivation induced protein breakdown to autophagy. In this study, we aimed to establish a cell-free assay recapitulating leucine-mediated autophagy in vitro and dissect its biochemical requirement. We found that in a cell-free assay, membrane association of Barkor/Atg14(L), a specific autophagosome-binding protein, is suppressed by cytosol from nutrient-rich medium and such suppression is released by nutrient deprivation. We also showed that rapamycin could efficiently reverse the suppression of nutrient rich cytosol, suggesting an essential role of mTORC1 in autophagy inhibition in this cell-free system. Furthermore, we demonstrated that leucine supplementation in the cultured cells blocks Barkor puncta formation and autophagy activity. Hence, we establish a novel cell-free assay recapitulating leucine-mediated autophagy inhibition in an mTORC1-dependent manner; this assay will help us to dissect the regulation of amino acids in autophagy and related human metabolic diseases.
补充支链氨基酸，特别是亮氨酸，对于通过调节蛋白质合成和降解来改善营养不良至关重要。新出现的证据将亮氨酸剥夺诱导的蛋白质降解与自噬联系起来。在这项研究中，我们旨在建立一种体外重现亮氨酸介导的自噬的无细胞测定法，并剖析其生化需求。我们发现，在无细胞测定中，Barkor/Atg14(L)，一种特定的自噬体结合蛋白，与富含营养的培养基中的细胞质的膜结合受到抑制，而这种抑制作用是由营养剥夺释放的。我们还表明，雷帕霉素可以有效地逆转富含营养的细胞质的抑制作用，表明 mTORC1 在该无细胞系统中自噬抑制中的重要作用。此外，我们证明在培养细胞中补充亮氨酸可阻止 Barkor 斑点的形成和自噬活性。因此，我们建立了一种新的无细胞测定法，可重现亮氨酸介导的、依赖于 mTORC1 的自噬抑制；该测定法将有助于我们剖析氨基酸在自噬和相关人类代谢疾病中的调控。
