Regulation of eukaryotic membrane structure and function

描述（由申请人提供）：由于质膜atp结合盒（ABC）转运蛋白的过度表达，在人类肿瘤中经常出现多药耐药。最近的实验表明，多药耐药细胞经常表现出鞘脂类的变化，同时ABC转运蛋白的表达和活性增加。我们最近发现，多重耐药酿酒酵母细胞在ABC转运蛋白表达的增加和鞘脂生物合成的变化之间表现出类似的分子联系。我们实验室和其他人的研究表明，锌簇转录因子pdr1p和Pdr3p活性的增加导致多药耐药ABC转运体如Pdr5p和Yor1p，以及长链碱基（LCB）转运体Rsb1p的诱导。lcb是真核细胞鞘脂生物合成的生物活性前体。我们假设ABC转运蛋白和LCB转运蛋白Rsb1p的协同调节是质膜正常调节和功能所必需的。酿酒酵母中的Ypk蛋白激酶被LCBs激活，我们将通过调控Pdr3p来研究Ypk2p控制多药耐药和基因表达的能力。Pdr5p的缺失导致LCB耐药的产生。将进行遗传分析，以确定和分析该信号通路的组成部分，该信号通路将ABC转运体的丢失与LCB耐受性的诱导联系起来。LCBs的水平也将在缺乏Pdr5p和Yor1p的细胞以及具有不同水平Pdr1p/Pdr3p转录活性的突变株中进行分析。这将使我们能够确定受Pdr通路功能调节的lcb的身份，并揭示可能用于调节Pdr1p和/或Pdr3p活性的鞘脂。最后，鞘脂前体的积累对ABC转运蛋白功能的影响将被确定。在暴露于高水平LCBs或含有鞘脂合成缺陷的菌株中，将测量转运蛋白活性和运输。我们的工作表明，耐药酵母细胞与这些转运体将发挥作用的膜成分共同调节药物转运体的生物合成。这种协调调节对于确保正常的膜功能很重要，并且可能允许新的抗真菌药物靶向其中断，因为该途径的后期阶段是由哺乳动物中未发现的酶进行的。

DESCRIPTION (provided by applicant): Multidrug resistance often arises in human tumors due to overexpression of plasma membrane ATP-binding cassette (ABC) transporter proteins. Recent experiments have indicated that multidrug resistant cells frequently exhibit changes in the profile of lipid species known as sphingolipids along with increases in ABC transporter expression and activity. We have recently discovered that multidrug resistant Saccharomyces cerevisiae cells exhibit a similar molecular linkage between increases in ABC transporter expression and changes in sphingolipid biosynthesis. Work from our lab and others has shown that increases in the activity of the zinc cluster transcription factors Pdr1 p and Pdr3p lead to induction of multidrug resistant ABC transporters like Pdr5p and Yor1p but also the long chain base (LCB) transporter Rsb1p. LCBs are bioactive precursors in sphingolipid biosynthesis in eukaryotic cells. We hypothesize that the coregulation of the ABC transporters and LCB transporter Rsb1p is required for normal modulation and function of the plasma membrane. The Ypk protein kinases in S. cerevisiae are activated by LCBs and we will examine the ability of Ypk2p to control multidrug resistance and gene expression through regulation of Pdr3p. Loss of Pdr5p leads to induction of LCB resistance. Genetic analysis will be performed to identify and analyze components of this signaling pathway connecting loss of this ABC transporter with induction of LCB tolerance. Levels of LCBs will also be analyzed in cells lacking Pdr5p and Yor1p as well as in mutant strains with different levels of Pdr1p/Pdr3p transcriptional activity. This will allow us to determine both the identity of LCBs that are regulated by Pdr pathway function and to uncover sphingolipids that may serve to regulate activity of Pdr1p and/or Pdr3p. Finally, the effect of accumulation of sphingolipid precursors on ABC transporter function will be determined. Transporter activity and trafficking will be measured in strains exposed to high levels of LCBs or containing defects in sphingolipid synthesis. Our work indicates that drug resistant yeast cells co-regulate the biosynthesis of drug transporters with constituents of the membranes in which these transporters will function. This coordinate regulation is important to ensure normal membrane function and may allow new antifungal drugs to be targeted towards its interruption as later stages of this pathway are carried out by enzymes that are not found in mammals.