Projekt A) Identification of the functions of ergosteryl-ß-glucoside in the degradation of peroxisomes in the yeast. Projekt D) Dissecting glucosylceramide biosynthesis in the yeast Pichia pastoris

项目 A) 鉴定麦角甾醇-α-葡萄糖苷在酵母中过氧化物酶体降解中的功能。

• 批准号: 145798870
• 负责人: Privatdozent Dr. Dirk Warnecke
• 金额: --
• 依托单位: Biozentrum Klein Flottbek und Botanischer Garten
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/145798870?language=en
• 关键词: Projekt; Identification; functions; ergosteryl; glucoside

Projekt A)Autophagy is a cellular, degenerative pathway which is conserved in eukaryotes. The yeast Pichia pastoris can degrade its peroxisomes by a specific autophagic process called micropexophagy. We showed that P. pastoris mutants which are either deficient in ergosteryl-β-glucoside biosynthesis or in the localization of the ergosterol-β-glucosyltransferase are impaired in micropexophagy. The aim of the project is to reveal whether the ergosterol-β-glucosyltransferase activity and its lipid product, ergosteryl-β-glucoside, are essential for micropexophagy or the protein itself. It might be that the protein supports micropexophagy independently of its ergosterol-β-glucosyltransferase activity e.g. by protein-protein interactions. Therefore, we expressed variants of the ergosterol-β-glucosyltransferase with single amino acid alterations within the catalytic domain in P. pastoris. These cells will be examined for ergosteryl-β-glucoside biosynthesis and micropexophagy. Since preliminary results suggest that the glycolipid is required for micropexophagy, it is interesting to determine the structural details of the ergosteryl-β-glucoside which are required to fulfill its function. Therefore, we will replace the native ergosteryl-β-glucoside with structurally similar glycolipids and determine whether they support proper micropexophagy. To reach this goal, sterol glycosyltransferases of different sugar specificity will be expressed in P. pastoris.Projekt D)Sphingolipids are ubiquitous membrane lipids present in all eukaryotic and some prokaryotic organisms. Most fungi contain two different types of glycosylated sphingolipids with a distinct ceramide backbone: Glucosylceramide (GlcCer) and glycosyl inositol phosphorylceramide (GIPC). Our longterm goal is to establish the yeast Pichia pastoris as a model system to investigate the spatial separation and intracellular trafficking of sphingolipids, the formation of lipid domains and the biological functions of these structurally different types of glycosphingolipids. We started our work by the identification and characterization of six genes and their corresponding enzymes which are involved in the introduction of functional groups into the ceramide backbone, such as desaturases, hydroxylases, methyltransferases and glycosyltransferases. Subsequently, knock-out mutants of P. pastoris, each impaired in the function of one of these genes, were generated. In order to achieve a complete description of GlcCer biosynthesis in P. pastoris, the free ceramides present in these mutant strains will now be analyzed by LC/MS. In addition, the so far hypothetical GIPCs from this yeast will be purified and identified by mass spectrometrical methods.

自噬是一种在真核生物中保守的细胞退化途径。毕赤酵母可以通过一种特殊的自噬过程降解过氧化物酶体，这种自噬过程被称为微噬。我们发现，缺乏麦角甾醇-β-葡萄糖苷生物合成或麦角甾醇-β-葡萄糖基转移酶定位的巴斯德毕赤酵母突变体在微体吞噬中受损。该项目的目的是揭示麦角甾醇-β-葡萄糖基转移酶活性及其脂质产物麦角甾醇-β-葡萄糖苷是否是微噬或蛋白质本身所必需的。可能是该蛋白质支持微粒自噬，而不依赖于其麦角固醇-β-葡糖基转移酶活性，例如通过蛋白质-蛋白质相互作用。因此，我们在巴斯德毕赤酵母中表达了在催化结构域内具有单个氨基酸改变的麦角固醇-β-葡糖基转移酶的变体。将检查这些细胞的麦角甾醇-β-葡萄糖苷生物合成和微噬作用。由于初步结果表明糖脂是微食所必需的，因此确定麦角甾醇-β-葡萄糖苷的结构细节是有趣的，这是实现其功能所必需的。因此，我们将用结构相似的糖脂替代天然麦角甾醇-β-葡萄糖苷，并确定它们是否支持适当的微吞噬。为了达到这一目标，将在巴斯德毕赤酵母中表达不同糖特异性的甾醇糖基转移酶。D）鞘脂是存在于所有真核生物和一些原核生物中的普遍存在的膜脂质。大多数真菌含有两种不同类型的具有不同神经酰胺骨架的糖基化鞘脂：葡萄糖神经酰胺（GlcCer）和糖基肌醇磷酸神经酰胺（GIPC）。我们的长期目标是建立一个酵母毕赤酵母模型系统来研究鞘脂的空间分离和胞内运输，脂质结构域的形成以及这些结构不同类型的鞘糖脂的生物学功能。我们开始我们的工作，通过鉴定和表征的六个基因和它们相应的酶，这些基因和酶参与将功能基团引入到神经酰胺骨架中，例如去饱和酶，羟化酶，甲基转移酶和糖基转移酶。随后，产生了巴斯德毕赤酵母的敲除突变体，每个突变体在这些基因之一的功能中受损。为了实现巴斯德毕赤酵母中GlcCer生物合成的完整描述，现在将通过LC/MS分析这些突变菌株中存在的游离神经酰胺。此外，将通过质谱方法纯化和鉴定迄今为止假设的来自该酵母的GIPC。