TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.
TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.
复制标题
TORC2依赖性蛋白激酶YPK1磷酸化神经酰胺合酶以刺激复杂的鞘脂的合成。
DOI：
10.7554/elife.03779
复制
发表时间：
2014-10-03
期刊：
影响因子：
7.7
通讯作者：
Thorner J
中科院分区：
文献类型：
--
作者：
Muir A;Ramachandran S;Roelants FM;Timmons G;Thorner J
质膜脂质组成必须在生长过程中和环境的侮辱下保持。在酵母中，由TOR复合物2（TORC 2）依赖性蛋白激酶Ypk 1介导的信号传导控制响应于膜应激的脂质丰度和分布。Ypk 1，除其他行动，消除了负调节L-丝氨酸：棕榈酰辅酶A酰基转移酶，上调生产的长链基地前体鞘脂。为了探索TORC 2-Ypk 1信号在膜稳态中的其他作用，我们设计了一个三层全基因组筛选来识别其他Ypk 1底物，该底物精确定位了神经酰胺合酶复合物的两个催化亚基。YPK 1依赖磷酸化的两种蛋白质增加后，无论是鞘脂耗竭或热休克，是重要的细胞存活。鞘脂组学、其他生化测量和遗传分析表明，神经酰胺合酶的这些修饰增加了其比活性，并刺激长链碱基前体通道转化为鞘脂终产物。在该分支点的控制还可以防止可能通过刺激自噬而损害细胞生长的中间产物的积累。DOI：http://dx.doi.org/10.7554/eLife.03779.001细胞被质膜包围，质膜将每个细胞与环境隔开并保护每个细胞。这些膜由各种蛋白质和称为脂质的脂肪分子组成，这些分子在整个膜中精心组织。当细胞经历诸如热或过度压力的压力时，质膜发生变化以帮助保护细胞。特别是，在应激条件下，更多的一组称为鞘脂的脂质被掺入膜中。在酵母细胞中，一种名为Ypk 1的蛋白质在保护细胞免受压力方面起着重要作用。YPK 1控制许多蛋白质的活性，这些蛋白质负责平衡细胞膜中不同类型脂质的量。这些Ypk 1依赖性蛋白的联合作用导致细胞膜的重塑以保护免受压力。虽然已知有几种蛋白质与Ypk 1一起起作用，但一些用于保护质膜的变化不能单独用这些蛋白质的作用来解释。为了更全面地了解Ypk 1如何帮助细胞对环境变化做出反应，Muir等人开发了一种新方法，该方法结合了生物化学，遗传学和生物信息学技术来调查酵母基因组中可能是Ypk 1靶点的蛋白质。Muir等人首先通过搜索与已知Ypk 1靶点具有相似特征的蛋白质，产生了一系列潜在的候选蛋白质，然后考虑了那些已知参与Ypk 1过程的蛋白质。为了进一步筛选潜在的靶点，Muir等人在酵母细胞中进行了实验，以观察哪些蛋白质在过度生产时会阻止正常细胞生长。进一步的实验研究了这些蛋白质中哪些蛋白质在纯化后与Ypk 1相互作用，确定了12种新的蛋白质，它们最有可能是Ypk 1蛋白质的靶点。这些新鉴定的Ypk 1靶蛋白中的两个形成了称为神经酰胺合酶的酶复合物的一部分，该酶复合物产生一个蜡质脂质分子家族，从该家族中构建出更复杂的鞘脂。Muir等人发现，在应激期间，Ypk 1增强神经酰胺合成酶的活性，从而增加脂质的产生和沉积在细胞膜上的鞘脂的量。如果这个过程在任何阶段被中断，细胞就会在压力条件下挣扎生存。Muir等人鉴定的其他候选蛋白仍有待验证并表征为Ypk 1靶标。尽管如此，所使用的技术已经确定了一些新的Ypk 1靶点，也可以应用于其他生物过程中靶向蛋白的类似搜索。DOI：http://dx.doi.org/10.7554/eLife.03779.002网站
Plasma membrane lipid composition must be maintained during growth and under environmental insult. In yeast, signaling mediated by TOR Complex 2 (TORC2)-dependent protein kinase Ypk1 controls lipid abundance and distribution in response to membrane stress. Ypk1, among other actions, alleviates negative regulation of L-serine:palmitoyl-CoA acyltransferase, upregulating production of long-chain base precursors to sphingolipids. To explore other roles for TORC2-Ypk1 signaling in membrane homeostasis, we devised a three-tiered genome-wide screen to identify additional Ypk1 substrates, which pinpointed both catalytic subunits of the ceramide synthase complex. Ypk1-dependent phosphorylation of both proteins increased upon either sphingolipid depletion or heat shock and was important for cell survival. Sphingolipidomics, other biochemical measurements and genetic analysis demonstrated that these modifications of ceramide synthase increased its specific activity and stimulated channeling of long-chain base precursors into sphingolipid end-products. Control at this branch point also prevents accumulation of intermediates that could compromise cell growth by stimulating autophagy. DOI: http://dx.doi.org/10.7554/eLife.03779.001 Cells are enclosed by a plasma membrane that separates and protects each cell from its environment. These membranes are made of a variety of proteins and fatty molecules called lipids, which are carefully organized throughout the membrane. When cells experience stresses such as heat or excessive pressure, the plasma membrane changes to help protect the cell. In particular, more of a group of lipids called sphingolipids are incorporated into the membrane under stress conditions. In yeast cells, a protein called Ypk1 plays an important role in protecting the cell from stress. Ypk1 controls the activity of a number of proteins that are responsible for balancing the amounts of different types of lipids in cell membranes. The combined action of these Ypk1-dependent proteins leads to the remodelling of the cell membrane to protect against stress. While several proteins that work with Ypk1 are known, some of the changes that serve to protect the plasma membrane cannot be explained by the action of these proteins alone. To provide a more comprehensive picture of how Ypk1 helps cells to respond to changes in the environment, Muir et al. developed a new approach that combines biochemical, genetic and bioinformatics techniques to survey the yeast genome for proteins that could be Ypk1 targets. Muir et al. first produced a list of potential candidate proteins by searching for proteins with features similar to known Ypk1 targets, and then considered those that are known to be involved in processes that also involve Ypk1. To filter the potential targets further, Muir et al. performed experiments in yeast cells to see which proteins prevented normal cell growth if they were over-produced. Further experiments investigating which of these proteins interact with Ypk1 when purified identified 12 new proteins that are most likely targets of the Ypk1 protein. Two of these newly identified Ypk1 target proteins form part of an enzyme complex called ceramide synthase, which produces a family of waxy lipid molecules from which more complex sphingolipids are built. Muir et al. discovered that during stress, Ypk1 enhances the activity of the ceramide synthase enzyme, which increases lipid production and the amount of sphingolipid deposited in the cell membrane. If this process is interrupted at any stage, cells struggle to survive under stress conditions. The other candidate proteins identified by Muir et al. remain to be validated and characterized as Ypk1 targets. Nevertheless, the techniques used have conclusively identified some new Ypk1 targets and could also be applied to similar searches for proteins targeted in other biological processes. DOI: http://dx.doi.org/10.7554/eLife.03779.002