Cold Adaptation in Yeast: The Role of ER-Associated Degradation and Sterol Metabolism

智力价值：地球上生命的故事很可能是一个关于寒冷的故事。地球上最早的有机分子和细胞可能是在冰冷的环境中出现的，这为生命的进化提供了一个“冷起点”。就在最近的5亿年前，生命不得不在全球冰河时期生存下来。即使在今天，地球生物圈大部分都是寒冷的，高达80%的平均温度低于5摄氏度。显然，了解生物如何适应寒冷的温度对许多学科都很重要，从生命的进化到北极环境的生态学，再到细胞生物学和生理学。然而，令人惊讶的是，在我们对冷适应生物学的探索中存在着巨大的差距。最值得注意的是，作为五大经典生命体之一，真菌对寒冷适应的研究几乎不存在。此外，对任何生物体中固醇代谢在冷适应中的作用的研究也同样罕见。本项目将进行的实验将为深入探索酵母（一种单细胞真菌）的遗传学、分子和细胞生物学以及冷适应生理学奠定基础。这些研究的结果很可能与其他真菌有关，也许与其他生命王国有关。在寻找酿酒酵母内质网生物生成所需的基因时，赖特实验室发现，参与内质网相关降解（ERAD）的基因子集发生突变，导致冷敏感性。在与Martin Bard博士（印第安纳大学-普渡大学印第安纳波利斯分校）的合作中，他们还发现这些基因是适当调节固醇代谢所必需的。这些观察结果导致了本项目将要进行的实验的基本假设：ERAD调节酵母中固醇代谢的关键方面，这种调节是冷适应所必需的。为了验证这一假设，pi将使用遗传、生化和细胞生物学方法来确定ERAD调节甾醇代谢的分子机制，以及这种调节是否构成ERAD本身在冷适应中的作用。为了研究甾醇代谢在寒冷适应中的生态和进化相关性，酿酒酵母的实验将与南极环境中分离的嗜冷酵母的甾醇分析相协调。此外，还将确定酿酒酵母中冷适应所必需的基因。因此，这些实验的结果将为研究甾醇代谢和ERAD在酵母冷适应中的作用提供具体的见解，并为酵母冷适应的长期研究创造一个框架。影响：实验方案对培养研究生和本科生具有正统的基础研究价值。至少一名研究生和六名本科生将在传统的实验室研究环境中参与这些实验。然而，这个项目也将作为新的教育策略的孵化器和熔炉，特别是将真实的研究经验纳入入门生物学实验室。例如，在一个项目中，入门生物学的学生团队将从各种南极酵母物种中克隆HMG-CoA还原酶（一种高度保守的甾醇生物合成酶）的基因，对这些基因进行测序，并利用这些信息来研究酵母的系统发育。这样的实验结果可能会被发表，这是“真实”研究的标志。由于目前没有标准来定义最适合教学实验室环境的项目类型，这些基于教学实验室的项目将被全面评估，以制定成功的基于教学实验室的研究项目的基准。这些信息将用于评估其他研究者的潜在项目。通过为教师提供将研究项目带入教学实验室的机会，这一努力将促进教师的研究和教育任务更广泛和富有成效的整合，为学生提供更好的学习体验，并促进科学的更快发展。

Intellectual Merit: The story of life on Earth is likely to be a story of cold. The first organic molecules and cells may have arisen on Earth in icy conditions, providing an evolutionary "cold start" to life. As recently as 500 million years ago, life has had to survive through global glaciations. Even today, Earth's biosphere is largely cold, with as much as 80% having an average temperature of less than 5oC. Clearly, understanding how organisms adapt to cold temperature is important for a variety of disciplines, from the evolution of life to the ecology of arctic environments to cellular biology and physiology. However, surprisingly large gaps exist in our exploration of the biology of cold adaptation. Most notably, investigations of cold adaptation are nearly non-existent in fungi, one of the 5 classical kingdoms of life. In addition, investigations of the roles of sterol metabolism in cold adaptation in any organism are similarly uncommon. The experiments that will be performed in this project will lay the foundations for deep exploration of the genetics, molecular and cellular biology, and physiology of cold adaptation in yeast, a unicellular fungus. It is likely that results of these studies will have relevance to other fungi, and perhaps to other kingdoms of life.In a search for genes required for ER biogenesis in the yeast Saccharomyces cerevisiae, the Wright lab discovered that mutations in a subset of genes involved in ER-associated degradation (ERAD) result in cold sensitivity. In collaboration with co-PI Dr. Martin Bard (Indiana University - Purdue University Indianapolis), they also discovered that these genes are required for proper regulation of sterol metabolism. These observations lead to the foundational hypothesis for the experiments to be performed in this project: ERAD regulates key aspects of sterol metabolism in yeast and this regulation is required for cold adaptation. To test this hypothesis, the PIs will use genetic, biochemical, and cell biological approaches to determine both the molecular mechanisms by which ERAD regulates sterol metabolism and also whether this regulation underlies the role of ERAD itself in cold adaptation. To examine the ecological and evolutionary relevance of sterol metabolism in cold adaptation, experiments in S. cerevisiae will be coordinated with analyses of sterols in psychrophilic yeast species isolated in Antarctic environments. In addition, the genes in S. cerevisiae that are necessary for cold adaptation will be determined. Thus, results of these experiments will provide specific insights into the role of sterol metabolism and ERAD in cold adaptation, as well as create a framework for long-term investigations of cold adaptation in yeast. Broader Impacts: The experimental plan has the orthodox value of basic research for training graduate and undergraduate students. At least one graduate student and six undergraduates will be involved in these experiments in traditional laboratory research contexts. However, this project will also serve as an incubator and crucible for novel educational strategies, specifically the incorporation of authentic research experiences into introductory biology laboratories. For example, in one project, student teams in introductory biology will clone the gene for HMG-CoA reductase (a highly conserved sterol biosynthetic enzyme) from a variety of Antarctic yeast species, sequence these genes, and use this information to study the phylogeny of the yeasts. Such experimental results would be potentially publishable, the hallmark of "authentic" research. Because no standards currently exist to define the types of projects most suited to the teaching-lab environment, these teaching laboratory-based projects will be comprehensively assessed to develop benchmarks for successful teaching-laboratory based research projects. This information will be used to evaluate potential projects from other investigators. By developing opportunities for faculty to bring research projects into teaching laboratories, this effort will promote more extensive and productive integration of the research and education missions of faculty, a better learning experience for students, and more rapid advance of science.