Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
Mol Biol Cell. 2019 Jun 1;30(12):1555-1574. doi: 10.1091/mbc.E18-10-0682. Epub 2019 Apr 10.
Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2--associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5-phosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.
真核细胞的存活需要维持质膜（PM）的内环境稳定，以应对环境胁迫和脂质代谢的变化。在酵母中，PM 内环境稳态的关键调节剂是雷帕霉素靶蛋白（TOR）复合物 2（TORC2），这是一个包含进化上保守的 TOR 蛋白激酶同工型 Tor2 的多蛋白复合物。PM 的定位对于 TORC2 的功能至关重要。一个核心的 TORC2 亚基（Avo1）和两个与 TORC2 相关的调节因子（Slm1 和 Slm2）含有pleckstrin 同源（PH）结构域，该结构域特异性结合磷脂酰肌醇-4,5-二磷酸（PtdIns4,5P2）。为了研究 PtdIns4,5P2 和 TORC2 组成亚基的作用，我们使用了生长素诱导降解系统地消除这些因素，然后检查了剩余 TORC2 成分的定位、结合和功能。我们发现 PtdIns4,5P2 的耗竭显著降低了 TORC2 的活性，但并没有阻止 PM 的定位或 TORC2 的解体。此外，截短的 Avo1（缺乏其 C 端 PH 结构域）仍然被招募到 PM 并支持生长。即使所有三个含有 PH 的蛋白都不存在，剩余的 TORC2 亚基仍与 PM 结合。值得注意的是，Avo3 独立于 Avo1 和 Tor2 定位于 PM，而 Tor2 和 Avo1 都需要 Avo3 才能锚定在 PM 上。我们的研究结果提供了关于 TORC2 的新的机制信息，并确定 Avo3 对于 TORC2 在体内的 PM 定位和组装是至关重要的。
