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，人类肿瘤中经常出现多药耐药性。最近的实验表明，多药耐药细胞经常表现出鞘脂类脂质谱的变化，以及 ABC 转运蛋白表达和活性的增加。我们最近发现，多重耐药酿酒酵母细胞在 ABC 转运蛋白表达增加和鞘脂生物合成变化之间表现出类似的分子联系。我们实验室和其他实验室的工作表明，锌簇转录因子 Pdr1 p 和 Pdr3p 活性的增加会导致诱导多药耐药 ABC 转运蛋白（如 Pdr5p 和 Yor1p）以及长链碱基 (LCB) 转运蛋白 Rsb1p。 LCB 是真核细胞鞘脂生物合成中的生物活性前体。我们假设 ABC 转运蛋白和 LCB 转运蛋白 Rsb1p 的共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 LCB 激活，我们将检查 Ypk2p 通过调节 Pdr3p 控制多药耐药性和基因表达的能力。 Pdr5p 的缺失会导致 LCB 耐药的诱导。将进行遗传分析来识别和分析该信号通路的组成部分，该通路将 ABC 转运蛋白的丢失与 LCB 耐受性的诱导联系起来。还将分析缺乏 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的 LCB 水平。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，将确定鞘脂前体的积累对 ABC 转运蛋白功能的影响。将在暴露于高水平 LCB 或含有鞘脂合成缺陷的菌株中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蛋白发挥作用的膜成分共同调节药物转运蛋白的生物合成。这种协调调节对于确保正常的膜功能非常重要，并且可能允许新的抗真菌药物针对其中断，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.