From Feast to Famine: Regulating carbon metabolism in yeast

葡萄糖是大多数生物体维持生命的首选碳源。酿酒酵母将葡萄糖转化为乙醇和二氧化碳来产生能量。当葡萄糖变得有限或缺乏时会发生什么？细胞适应。当葡萄糖耗尽时，酵母可以使用乙醇作为替代碳源，从而使细胞得以存活。然而，乙醇浓度的增加会对细胞过程产生负面影响，并可能对细胞产生毒性。在葡萄酒、啤酒和生物乙醇生产过程中进行重要商业酒精发酵的酵母通常会经历葡萄糖耗尽和酒精水平上升的过程。这些条件会影响酵母的生理和新陈代谢，最终降低其发酵效率。了解酵母对环境中葡萄糖耗竭和乙醇的反应，将为碳代谢调节的基本过程提供新的见解，对乙醇生产行业具有潜在的好处。 在丰富的葡萄糖环境中，酵母菌发酵生长所需的基因被活跃地表达，而那些在没有葡萄糖的情况下利用非发酵碳源(如乙醇)生长所需的基因被抑制；这被称为葡萄糖抑制。当葡萄糖耗尽，乙醇是唯一的碳源时，这种转录调控就会逆转。同样，蛋白质的丰度、活性和细胞内的运输也可以受到普遍存在的碳条件的调节。这些适应性转录和/或蛋白质调节反应受信号机制的控制。尽管已经确定了几种调控蛋白和调控酵母适应葡萄糖耗竭的机制，但这些调控机制的几个重要方面仍然没有解决。我们将使用适应性转录和蛋白质调节的例子作为一种手段来研究新的调节蛋白质在碳信号中的功能。 Vid30蛋白复合体(Vid30c)是一种已知的E3泛素连接酶，它将一个小分子泛素连接到目标蛋白，最终导致目标蛋白的降解。我们最近发现，Vid30c对于葡萄糖耗竭的分子适应是必需的。除了已知的参与己糖转运蛋白Hxt3的降解外，当乙醇是唯一的碳源时，Vid30c也是葡萄糖抑制基因转录激活所必需的。Vid30c用来调节乙醇中这些特定事件的分子和调控机制目前尚不清楚。这项研究的目的是阐明Vid30c在酵母适应乙醇过程中的作用机制。我们将使用酵母遗传、分子和细胞生物学方法来鉴定和表征特定的蛋白质，包括调节蛋白和Vid30c的直接靶标，这些蛋白质介导依赖Vid30c的葡萄糖抑制基因的激活和Hxt3在酒精适应过程中的降解。此外，我们还将确定决定Vid30c在乙醇中的功能特异性的特定蛋白质。这项拟议的研究将为酵母中碳调节的一个目前知之甚少的方面提供新的基本见解。解决酵母菌对葡萄糖耗尽和增加乙醇条件的分子适应问题，可以导致开发提高这种具有重要商业价值的生物的发酵效率的策略。

Glucose is the preferred carbon source that supports life in most organisms. The yeast Saccharomyces cerevisiae converts glucose into ethanol and carbon dioxide to generate energy. What happens when glucose becomes limiting or is absent? Cells adapt. Yeast can use ethanol as an alternative carbon source when glucose is depleted, thereby allowing the cell to survive. However, increasing concentrations of ethanol negatively impacts cellular processes and can become toxic to the cell. Yeast performing commercially important alcoholic fermentations during wine, beer and bio-ethanol production commonly experience glucose depletion and increasing levels of ethanol as the fermentation proceeds. These conditions influence the yeast’s physiology and metabolism that ultimately decrease its fermentation efficiency. Understanding the response of yeast to glucose depletion and ethanol in its environment will provide novel insight into the fundamental process of carbon metabolism regulation with potential benefits to ethanol-producing industries. In an abundant glucose environment the yeast genes needed for fermentative growth are actively expressed, while those needed for growth in the absence of glucose using a non-fermentable carbon source, like ethanol, are inhibited; this is known as glucose repression. This transcriptional regulation reverses when glucose is depleted and ethanol is the sole carbon source. Similarly, protein abundance, activity and intracellular trafficking can also be regulated by the prevailing carbon conditions. These adaptive transcription and/or protein regulation responses are governed by signaling mechanisms. Despite the identification of several regulatory proteins and mechanisms that govern the adaptation of yeast to glucose depletion, several important aspects of these regulatory mechanisms remain unresolved. We will use examples of adaptive transcription and protein regulation as a means to study the function of new regulatory proteins in carbon signaling. The Vid30 protein complex (Vid30c) is an E3 ubiquitin ligase known to link a small molecule, ubiquitin, to target proteins that ultimately result in the degradation of the target protein. We showed recently that the Vid30c was needed for the molecular adaptation to glucose depletion. In addition to its known involvement in the degradation of the hexose transporter Hxt3, the Vid30c is also needed for the transcriptional activation of glucose repressed genes when ethanol is the sole carbon source. The molecular and regulatory mechanisms used by the Vid30c to mediate these specific events in ethanol are currently unknown. The goal of the proposed research is to delineate the mechanisms of function of the Vid30c during the yeast’s adaptation to ethanol. We will use a combination of yeast genetic, molecular and cellular biology approaches to identify and characterize specific proteins, including regulatory proteins and direct targets of the Vid30c, that mediate the Vid30c-dependent activation of glucose-repressed genes and the degradation of Hxt3 during the adaptation to ethanol. In addition, we will identify specific proteins that determine the functional specificity of the Vid30c in ethanol. The proposed research will provide novel fundamental insight into an aspect of carbon regulation in yeast that is currently poorly understood. Resolving the molecular adaptation of yeasts to glucose depletion and increasing ethanol conditions can lead to the development of strategies to increase the fermentation efficiency of this commercially important organism.