Stjepanovic Goran, Baskaran Sulochanadevi, Lin Mary G, Hurley James H
Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Mol Cell. 2017 Aug 3;67(3):528-534.e3. doi: 10.1016/j.molcel.2017.07.003. Epub 2017 Jul 27.
The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is required for the initiation of essentially all macroautophagic processes. PI3KC3-C1 consists of the lipid kinase catalytic subunit VPS34, the VPS15 scaffold, and the regulatory BECN1 and ATG14 subunits. The VPS34 catalytic domain and BECN1:ATG14 subcomplex do not touch, and it is unclear how allosteric signals are transmitted to VPS34. We used EM and crosslinking mass spectrometry to dissect five conformational substates of the complex, including one in which the VPS34 catalytic domain is dislodged from the complex but remains tethered by an intrinsically disordered linker. A "leashed" construct prevented dislodging without interfering with the other conformations, blocked enzyme activity in vitro, and blocked autophagy induction in yeast cells. This pinpoints the dislodging and tethering of the VPS34 catalytic domain, and its regulation by VPS15, as a master allosteric switch in autophagy induction.
III类磷脂酰肌醇3激酶复合物I（PI3KC3-C1）是几乎所有巨自噬过程起始所必需的。PI3KC3-C1由脂质激酶催化亚基VPS34、VPS15支架以及调节性亚基BECN1和ATG14组成。VPS34催化结构域与BECN1:ATG14亚复合物不接触，目前尚不清楚变构信号是如何传递给VPS34的。我们利用电子显微镜和交联质谱分析来剖析该复合物的五种构象亚态，其中一种构象亚态是VPS34催化结构域从复合物中脱离，但仍通过一个内在无序的连接子相连。一种“拴系”构建体可防止脱离，同时不干扰其他构象，在体外阻断酶活性，并在酵母细胞中阻断自噬诱导。这确定了VPS34催化结构域的脱离和拴系及其受VPS15的调控，是自噬诱导中的一个主要变构开关。
