The ability to adapt to stress is one of the main characteristics of life. The molecular mechanisms by which such adaptation is achieved are of fundamental interest to modern biology. It is increasingly clear that stress tolerance is coupled to the cell's ability to be a good shepherd of its resources. The vital importance of molecular resource-managing systems is reflected in the remarkable evolutionary conservation of their key regulatory components. Members of the Snf1/AMP-activated protein kinase (AMPK) family are found in eukaryotes as complex as mammals and as simple as yeast. Mammalian AMPK is activated in response to energy limitation (increased cellular AMP-to-ATP ratios), and functions to restore the energy balance by stimulating ATP generation and inhibiting ATP consumption. Snf1 protein kinase of budding yeast (Saccharomyces cerevisiae) becomes activated and promotes the utilization of alternative carbon/energy sources when the preferred source, glucose, becomes limiting. In addition to glucose limitation, Snf1 has been implicated in responses to various other stress conditions. Due to advantages offered by the yeast system, studies of Snf1 have been providing invaluable insights into the most fundamental aspects of Snf1/AMPK structure and function. However, many questions still remain unanswered. It is known that Snf1 is activated by partially redundant upstream kinases (Sak1, Tos3, and Elm1) and inhibited by the Reg1-Glc7 phosphatase, but the exact mechanisms that shift the balance between these opposing processes remain unknown. To gain further knowledge, an innovative genetic screen for mutants with defects in the Snf1 pathway has been devised and performed. This project uses genetic, molecular, and biochemical methods to determine how the novel regulators thus identified interface with the known elements of the Snf1 pathway, and how they deliver signals from other sensory systems of the cell. This research will advance our understanding of the Snf1/AMPK family and its functions, general mechanisms of protein kinase regulation, and mechanisms of stress tolerance in eukaryotes. The results obtained will be of general interest to the scientific community. Broader impacts: Graduate and undergraduate students, including women and minorities, will participate in this project, and will receive excellent training to pursue their careers in research and science education. Experiments from this work will be incorporated into an experimental microbiology course for graduate and undergraduate students. The results obtained will be used to constantly update an advanced graduate course on signal transduction. Because Snf1-related kinases are involved in stress responses in plants, this work also has the potential to suggest new approaches to improving agricultural practices.
If living organisms were unable to manage stress, there would be no life on Earth. Stress can be generally defined as adverse conditions and the negative effects they produce on an organism. The molecular machinery of stress management is so important and ancient that its central pieces are still present in a wide array of species. Forms of the AMP-activated protein kinase (AMPK) are found in eukaryotic organisms as simple as yeast and as complex as humans. The primary role of AMPK is to manage stress caused by energy shortage. Under energy stress conditions, AMPK "wakes up" and instructs various cellular systems to balance the energy "budget" in two ways: (1) by reducing energy spending, and (2) by increasing energy "income". In humans, for example, AMPK helps stimulate starving cells to absorb more glucose from the bloodstream; defects in this process lead to type 2 diabetes, a disease in which cells dangerously head toward starvation while creating a dangerous buildup of "unclaimed" blood glucose, which can cause tissue and organ damage. While the general purpose of AMPK is clear, many facets of its function are not completely understood. For example, where does AMPK collect all of its information regarding energy stress? Although AMPK can directly detect the presence of "spent" energy molecules ("engine exhaust") such as AMP or ADP, there are other nutrient-sensing systems in the cell, and it would be extremely odd if AMPK did not "listen" to some of them. In other words, what is the complete list of AMPK’s information sources? A deeper understanding of these and other aspects will benefit society on multiple fronts – from fighting diabetes and obesity to improving stress tolerance and performance of yeast cells used for biofuel (ethanol) production. It would be fair to say that AMPK is a "molecule of national interest" and its studies are of profound importance. When studying the most fundamental properties of life, researchers often use so-called "model organisms". Budding yeast, also known as baker’s yeast and brewer’s yeast for its use in bread- and beer-making (and ethanol production), is one of the most popular model organisms. The advantages of yeast to researchers include its quick growth, its safety, and the lack of ethical concerns associated with laboratory animal research. In addition, AMPK was first discovered in yeast. For historical reasons, yeast researchers call this molecule SNF1 instead of AMPK. Since the discovery of SNF1/AMPK, budding yeast has served as a powerful model for understanding the most fundamental principles of its function. The main goal of this project was to gain a deeper insight into SNF1/AMPK taking advantage of budding yeast. In accord with questions raised above, the project aimed at a better understanding of the information inputs that SNF1 receives. First, improved methodology was developed to more accurately assess SNF1’s "on" and "off" states. This created a foundation for subsequent studies to better understand how SNF1 responds to signals from various sources. One of our studies implicated SNF1 in "listening in" on signals from protein kinase A, which is another nutrient-sensing system. Another study implicated the mitochondria as SNF1’s additional source of information, and the manner in which the mitochondria "talk" to SNF1 turned out to be entirely unexpected from the conventional standpoint. In another study, SNF1 was implicated in responses to nitrogen limitation, a stress that is not directly related to energy limitation, further emphasizing a broader role of SNF1 in stress management. Many important signals in the cell are relayed between molecules through direct "body-to-body" contact. Thus, understanding how SNF1 integrates its information is unthinkable without characterizing its interaction partners. The "classical" ways to study such partners (genetically or biochemically) generate evidence that is often not sufficiently direct, as nothing compares to being able to directly "see" molecules interact in a living cell. Therefore, our project included an interdisciplinary collaboration with physicists specializing in molecular imaging. This collaboration resulted in the development of a new and improved imaging approach for analyzing physical interactions between molecules inside live cells. Collaboration on developing new molecular imaging methodology is a strategically important investment for our cause. Besides publications in peer-reviewed journals (with two additional articles currently in revision), results from this project were disseminated through multiple presentations at local, national and international conferences. The project also served as an excellent platform for improving undergraduate and graduate education. Over 1000 students in undergraduate and graduate courses were exposed to the results, materials, and ideas generated in this project, and a peer-reviewed article was published in a journal specializing in biology education. Eight undergraduate students gained substantial research experience by conducting experiments on various aspects of the project. Six graduate students – including women and minorities - received advanced degrees (MS and PhD), and went on to have successful careers in areas such as science and education. .
