Molecular Genetic Analysis of Yeast Sphingolipids

描述(由申请人提供)：鞘磷脂正在成为膜组织和功能的关键元素，并作为调节压力保护和控制生长的信号转导途径的调节器。这些角色中的许多似乎在酵母和哺乳动物之间是保守的。在了解鞘磷脂在膜和信号中的功能方面已经取得了进展，但我们的知识仍然处于初级状态，需要付出大量努力才能实现更彻底的理解。我们以前的研究表明，鞘磷脂是酿酒酵母细胞生长所必需的，并在热应激中发挥作用。在这些研究的基础上，将实现三个具体目标。第一个目的是确定为什么在热应激时鞘磷脂是胞吐所必需的。具体地说，要确定哪种鞘脂是必需的，哪一步需要鞘脂，以及所需鞘脂的功能是什么。第二个目的是研究受狮身人面像长链碱基调控的可能的蛋白激酶。Pkh 1p和Pkh2p是哺乳动物磷酸肌醇依赖性蛋白激酶(PDKs)的同源物，Yck 2p是哺乳动物酪蛋白激酶的同源物。这些蛋白质调控酵母细胞生长和胁迫保护所必需的特性不佳的信号通路。此外，将使用一种新开发的蛋白质芯片技术筛选酵母蛋白质组，寻找与特定鞘脂结合的蛋白质，以确定需要鞘脂发挥功能的新信号通路和细胞过程。第三个目的是确定为什么鞘磷脂的超长链脂肪酸(VLCFA，C26)成分是液泡(溶酶体)被液泡ATPase酸化所必需的。VLCFA存在于所有真核生物中，但其功能尚不清楚。这些研究将揭示鞘磷脂在膜功能中的新作用，如在脂筏中，以及在胁迫保护、生长控制和必要的生理过程中至关重要的信号通路中。拟议的研究结果将有助于理解哺乳动物的信号通路，并有助于揭示药物干预包括癌症、动脉粥样硬化和神经疾病在内的人类疾病的有用靶点。

DESCRIPTION (provided by applicant): Sphingolipids are emerging as key elements in membrane organization and function and as regulators of signal transduction pathways that mediate stress protection and control growth. Many of these roles seem to be conserved between yeast and mammals. Progress has been made in understanding the functions of sphingolipids in membranes and in signaling but our knowledge remains rudimentary and much effort will be required to achieve a more thorough understanding. Our previous studies demonstrated that sphingolipids are essential for growth of Saccharomyces cerevisiae cells and that they play roles in heat stress. Three Specific Aims will build upon these studies. The first aim is to determine why sphingolipids are necessary for exocytosis during heat stress. Specifically, to determine which sphingolipid is necessary, which step requires sphingolipids, and what the function of the required sphingolipid is. The second aim is to characterize putative protein kinases regulated by sphingoid long chain bases. The protein kinases are Pkh 1p and Pkh2p, homologs of mammalian phosphoinositide-dependent kinases (PDKs), and Yck2p, a homolog of mammalian casein kinases. These proteins regulate poorly characterized signaling pathways necessary for yeast cell growth and stress protection. In addition, the yeast proteome will be screened, using a newly developed protein chip technique, for proteins that bind specific sphingolipids in order to identify novel signaling pathways and cellular processes that require sphingolipids for function. The third aim is to determine why the very long chain-fatty acid (VLCFA, C26) component of sphingolipids is necessary for acidification of vacuoles (lysosomes) by the vacuolar ATPase. VLCFAs are present in all eucaryotes but their functions remain unknown. These studies will reveal new roles for sphingolipids in membrane function, such as in lipid rafts, and in signaling pathways that are crucial for stress protection, growth control and essential physiological processes. The results of the proposed studies should aid in understanding mammalian signaling pathways and help to reveal useful targets for pharmacological intervention in human ailments including cancer, atherosclerosis and neurological disorders.