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

知识价值：地球上生命的故事很可能是一个寒冷的故事。第一个有机分子和细胞可能是在地球上冰冷的条件下出现的，为生命的进化提供了一个“冷启动”。就在5亿年前，生命不得不在全球冰川中生存。即使在今天，地球的生物圈大部分是寒冷的，多达80%的平均温度低于5摄氏度。很明显，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巨大的差距。最值得注意的是，在真菌中几乎不存在冷适应的研究，真菌是5个经典的生命王国之一。此外，研究固醇代谢在任何生物体冷适应中的作用也同样罕见。该项目的实验将为深入探索单细胞真菌酵母的遗传学、分子和细胞生物学以及低温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。在寻找酿酒酵母中ER生物合成所需的基因时，Wright实验室发现，参与ER相关降解（ERAD）的基因子集的突变导致冷敏感性。在与共同PI博士马丁巴德（印第安纳州大学-普渡大学印第安纳波利斯）合作，他们还发现，这些基因是必要的适当调节固醇代谢。这些观察结果导致在该项目中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，并且这种调节是冷适应所需的。为了验证这一假设，PI将使用遗传，生物化学和细胞生物学方法来确定ERAD调节固醇代谢的分子机制，以及这种调节是否是ERAD本身在冷适应中的作用的基础。为了研究冷适应过程中甾醇代谢的生态学和进化相关性，酿酒酵母的研究将与南极环境中分离的嗜冷酵母物种中甾醇的分析相协调。此外，S.将确定冷适应所必需的酿酒酵母。因此，这些实验的结果将提供具体的见解固醇代谢和ERAD在冷适应中的作用，以及创建一个框架，长期调查的冷适应酵母。 更广泛的影响：实验计划具有培养研究生和本科生的基础研究的正统价值。至少有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，这个项目也将作为一个孵化器和坩埚新的教育策略，特别是真实的研究经验纳入介绍生物学实验室。例如，在一个项目中，生物学导论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，并使用这些信息研究酵母的遗传。这样的实验结果可能是可验证的，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，这些教学实验室为基础的项目将进行全面评估，以制定成功的教学实验室为基础的研究项目的基准。这些信息将用于评估其他研究者的潜在项目。通过为教师开发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的更广泛和更富有成效的整合，为学生提供更好的学习体验，以及更快的科学进步。

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.