Role of Cdc48p and Msp1p in Peroxisomal Protein Quality Control and Communication

过氧化物酶体是单一的膜结合细胞器，非常特殊，因为1)它们输入折叠的，甚至低聚的蛋白质，2)这种输入是由在细胞质和过氧化物酶体膜之间循环的受体完成的。大多数过氧化物酶体基质酶由输入受体Pex5p输入。Pex5p受体周期的中心步骤包括细胞质中新合成的蛋白质的识别，然后是受体/货物与过氧化物酶体膜上的进口蛋白的组装。在货物释放后，Pex5p被单泛素化，并通过aaa型atp酶Pex1p和Pex6p以atp依赖的方式从膜上释放出来。受体回收中的缺陷会导致进口机器的阻塞。通过质量控制系统清除积聚受体的过氧化物酶体膜，可以避免这种情况。这个系统被称为RADAR（受体积累和降解在缺乏循环），在物种中是保守的，并导致受体从膜中提取及其蛋白酶体降解。然而，我们对RADAR通路的了解很少。在这个项目中，我们的目标是通过使用面包师酵母作为模式生物来确定RADAR途径所需的因素以及其他过氧化物的质量控制。为此，我们将追求以下主要目标：1)基于我们的初步数据表明，aaa - atp酶Cdc48p在这一过程中起主要作用，本项目的第一个目标是阐明Cdc48p在过氧化物酶体功能和维持中的分子功能。我们将研究雷达是否需要已知的Cdc48p接头蛋白。我们将在体外重建出口过程，并确定关键氨基酸残基，重点关注泛素化的作用。最后，我们将分离和表征过氧化物酶体和细胞质雷达相关机制。2)该项目的第二个目标是解决aaa - atp酶Msp1p，它以其在线粒体和过氧化物酶体尾锚定膜蛋白的质量控制中的作用而闻名。我们的初步数据表明，Msp1p的底物谱不仅限于尾部锚定蛋白，还包括其他类型的整体膜蛋白，包括具有多个膜跨度的蛋白。在这里，我们打算采用系统的方法来鉴定新的mspsp1靶点，从而研究Msp1是否在过氧化物酶体膜蛋白的质量控制中发挥更普遍的作用。3)根据我们的观察，过氧化物酶体和线粒体在非功能性Msp1p过表达时聚集，我们打算分离和表征相应的细胞器系。

Peroxisomes are single membrane-bound cell organelles that are very special as 1) they import folded, even oligomeric proteins and 2) this import is performed by receptors that cycle between the cytosol and the peroxisomal membrane. Most peroxisomal matrix enzymes are imported by the import receptor Pex5p. Central steps in the receptor cycle of Pex5p comprise the recognition of newly synthesized proteins in the cytosol followed by receptor/cargo assembly with the importomer at the peroxisomal membrane. Following cargo release, Pex5p is monoubiquitinated and released from the membrane by the AAA-type ATPases Pex1p and Pex6p in an ATP-dependent manner. A defect in receptor recycling would result in a block of the import machinery. This is avoided by a quality-control system that clears the peroxisome membrane of accumulating receptors. This system, termed RADAR (Receptor Accumulation and Degradation in Absence of Recycling), is conserved among species, and leads to the extraction of the receptor from the membrane and its proteasomal degradation. However, our knowledge on the RADAR pathway is scarce. In this project, we aim to identify the factors that are required for the RADAR pathway as well as the quality control of other peroxins by using baker’s yeast as a model organism. To this end, we will pursue the following main goals: 1) Based on our preliminary data demonstrating that the AAA-ATPase Cdc48p plays a major role in this process, the first goal of this project is to elucidate the molecular function of Cdc48p for peroxisome function and maintenance. We will investigate whether known Cdc48p adaptor proteins are required for RADAR. We will reconstitute the export process in vitro and identify crucial amino acid residues with focus on the role of ubiquitination. Finally, we will isolate and characterize the peroxisomal and cytosolic RADAR-associated machineries. 2) The second goal of this project addresses the AAA-ATPase Msp1p, which is known for its role in the quality control of mitochondrial and peroxisomal tail-anchored membrane proteins. Our initial data indicate that the substrate spectrum of Msp1p is not limited to tail-anchored proteins but also comprises other kind of integral membrane proteins, including proteins with multiple membrane spans. Here we intend to apply systematic approaches to identify novel Msp1p-targets and thus to investigate whether Msp1 performs a more general role in the quality control of peroxisomal membrane proteins. 3) Based on our observation that peroxisomes and mitochondria cluster upon overexpression of non-functional Msp1p, we intend to isolate and characterize the corresponding organelle tethers.