@article{c9140ce760544c9c80f4f9f08b37730a,
title = "Reconstitution of leucine-mediated autophagy via the mTORC1-Barkor pathway in vitro",
abstract = "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.",
keywords = "Autophagosome, Autophagy, Barkor/Atg14(L), LC3, P62, PtdIns3K/Vps34, Rapamycin, Rubicon, S6K",
author = "Xianghua Yan and Qiming Sun and An Ji and Yaqin Zhu and Zhengfei Liu and Qing Zhong",
note = "Funding Information: collected by centrifugation, washed three times with PBS and We thank all of the Zhong laboratory members for helpful resuspended in 20 ml of hypotonic buffer [20 mM TRIS-HCl discussion and technical assistance. This work was supported (pH 7.6), 5 mM KCl; 2 mM MgCl2, 0.5% NP-40] supple-by University of California Cancer Research Coordinating mented with protease inhibitors cocktail (Roche, 04693124001). Committee funds, a New Investigator Award for Aging from the The resuspended cells were lysed by treating (20 times) with Ellison Medical Foundation, Hellman Family Fund, American Dounce cell homogenizer and centrifuged at 12000 g for 15 min. Cancer Society grant (RSG-11-274-01-CCG) and NIH RO1 Both supernatant and the pellet were collected. The pelleted (CA133228) (to Q.Z.) and by the grants from the National Natural fraction was treated with 16 ml of high salt buffer [20 mM TRIS-Science Foundation of China (No.31172290, No.31072036 and HCl (pH 7.6), 450 mM NaCl, 0.1% NP-40, 25% glycerol, No.30700580 to X.Y.) and Huazhong Agricultural University 100 U DNase I/ml, protease inhibitor cocktail] and vortexed Scientific and Technological Self-innovation Foundation (No. vigorously and then centrifuged at 12000 g for 20 min. The 2010PY011 to X.Y.).",
year = "2012",
month = feb,
doi = "10.4161/auto.8.2.18563",
language = "English (US)",
volume = "8",
journal = "Autophagy",
issn = "1554-8627",
publisher = "Landes Bioscience",
number = "2",
}