TARGETING OF PROTEINS INTO PEROXISOMES

过氧化物体是普遍存在的亚细胞细胞器，密切参与 真核细胞中的多种代谢途径。减损 过氧化体蛋白的输入和生物发生是许多人类 过氧化物症(Zellweger综合征、高脂血酸血症、 婴儿Refsum病和新生儿肾上腺脑白质营养不良) 非常虚弱和致命的。我们的长期利益在于理解 基质和膜蛋白的运输机制 它们的合成部位，在细胞质中，到过氧化物体。其中两个 过氧化体靶向信号，pts1和pts2，参与了 已经确定了过氧化体基质蛋白和PTS1受体。 并以此为特征。然而，其他细胞质和细胞膜成分 目前还不清楚是否参与了过氧化体蛋白的输入。我们希望 使用生化和遗传策略的组合来识别 此导入过程中涉及的其他组件。这些研究将使 关于一种重要的亚细胞器的生物发生，以及 也与上面提到的人类疾病有关。的具体目标 该提案如下所示。 1.研究巴氏酵母PAS8蛋白作为PTS1受体的作用 2.体外通过PTS1途径定量检测过氧化物体的导入 3.过氧化物酶体所需酵母胞浆因子的鉴定 进口 4.建立可供选择的依赖胞质的体外进口系统 酵母 5.研究过氧化物酶体膜的靶向性和拓扑学 蛋白质类 6.研究巴斯德毕赤酵母PAS1和PAS5蛋白在大肠杆菌中的作用。 过氧化物体的生物发生

Peroxisomes are ubiquitous subcellular organelles intimately involved in multiple metabolic pathways in eukaryotic cells. The impairment of peroxisomal protein import and biogenesis is responsible for many human peroxisomal disorders (Zellweger's syndrome, Hyperpipecolic acidaemia, infantile Refsum's disease and neonatal adrenoleukodystrophy) which can be very debilitating and lethal. Our long term interest is in understanding the mechanism by which matrix and membrane proteins are transported from their site of synthesis, in the cytosol, to the peroxisomes. Two of the peroxisomal targeting signals, PTS1 and PTS2, involved in the import of peroxisomal matrix proteins, and the PTS1 receptor, have been identified and characterized. However, the other cytosolic and membrane components involved in peroxisomal protein import are unknown at present. We hope to use a combination of biochemical and genetic strategies to identify the other components involved in this import process. These studies will shed light on the biogenesis of an important subcellular organelle, and are also relevant to the human disorders mentioned above. The specific aims of the proposal are listed below. 1. To study the role of the P. pastoris PAS8 protein as the PTS1 receptor 2. Quantitation of peroxisomal import in vitro via the PTS1 pathway 3. Identification of yeast cytosolic factors required for peroxisomal import 4. Development of alternative cytosol-dependent in vitro import systems in yeast 5. To investigate the targeting and topology of peroxisomal membrane proteins 6. To study the role of the P. pastoris PAS1 and PAS5 proteins in peroxisome biogenesis