Mechanism of targeting of Peroxisome-Mitochondria localizing proteins

过氧化物酶体和线粒体是协同代谢细胞器，具有从脂质代谢到先天免疫应答的多种功能。这在一定程度上是由于共享调节它们形态和信号通路的基因。虽然只发现了少数，但这两种细胞器的密切关系表明，可能有更多的这种双过氧化物酶体-线粒体蛋白（dpmp）。因此，鉴定新的dpmp可能阐明这些细胞器在细胞过程中的新作用。在过去的十年里，我的nserc资助的研究项目集中在了解过氧化物酶体生物发生的分子机制。我们发现过氧化物酶体生物发生因子PEX16最初以内质网为靶点，在内质网招募其他过氧化物酶体膜蛋白，这些蛋白随后转运到过氧化物酶体。我们现在建议在我们的发现的基础上研究dpmp是如何靶向过氧化物酶体的。dpmp是一组功能多样的膜蛋白，定位于过氧化物酶体和线粒体。然而，这些膜蛋白如何能够靶向具有不同蛋白质进口机制的两种细胞器尚不清楚。我们的主要目标是了解dpmp是如何靶向过氧化物酶体的。过氧化物酶体膜蛋白（pmp）可以通过两种不同的途径，直接进入过氧化物酶体或通过内质网间接进入。我们最近表明，过氧化物酶体生物发生因子PEX16是间接途径所必需的，因为它可以将pmp招募到内质网。在我们未发表的工作中，我们发现PEX16可以在内质网上呈现多种拓扑结构，包括两跨膜和四跨膜蛋白。我们还发现，要将至少一种称为USP30的dPMP导入到ER， PEX16是必要和充分的。因此，我们假设PEX16通过首先将非经典pmp（如dpmp）导入ER介导其靶向。本研究的目的是：1。确定双定位蛋白（dpmp）是否通过PEX16导入过氧化物酶体；2. 识别dpmp对过氧化物酶体靶向的靶向信号；3. 用低温电镜测定PEX16的蛋白质结构。为了实现这些目标，我们将使用活细胞超分辨率显微镜与生化和分子生物学技术的结合。我们还将使用最新的冷冻电子显微镜来阐明PEX16的蛋白质结构。蛋白质靶向对正常的细胞功能至关重要。我们提出的研究处于过氧化物酶体蛋白靶向研究的前沿，并将为这种未被充分研究的细胞器如何招募蛋白质提供详细的见解。此外，我们从NSERC资助的研究中获得的知识可以被更广泛的生物界应用，以更广泛地了解过氧化物酶体及其在大量生理过程中的作用。

Peroxisomes and mitochondria are synergistic metabolic organelles sharing several functions from lipid metabolism to innate immune response. This is partly made possible by the sharing of genes that regulate their morphology and signaling pathways. Although only a handful has been discovered, the close relationship of the two organelles suggests there are likely more of these dual peroxisome-mitochondria proteins (dPMPs). Therefore, identifying novel dPMPs may elucidate new roles of these organelles in cellular processes. Over the last decade, my NSERC-funded research program has focused on understanding the molecular mechanisms of peroxisome biogenesis. We discovered that the peroxisomal biogenesis factor PEX16 is initially targeted to the ER where it recruits other peroxisomal membrane proteins that traffic subsequently to peroxisomes. We now propose to build upon our findings to study how dPMPs are targeted to peroxisomes. dPMPs are a functionally diverse group of membrane proteins that localize to both peroxisomes and mitochondria. However, how these membrane proteins are capable of targeting to both organelles that possess distinct protein import machinery is not known. Our main objective is to understand how dPMPs are targeted to peroxisomes. Peroxisomal membrane proteins (PMPs) can be imported by two distinct pathways, directly to peroxisomes or indirectly via the ER. We have recently shown that the peroxisomal biogenesis factor, PEX16, is required for the indirect pathway as it can recruit PMPs to the ER. In our unpublished work, we have found that PEX16 can take on multiple topologies on the ER including two-transmembrane and four-transmembrane proteins. We have also found that PEX16 is necessary and sufficient to import at least one dPMP called USP30 to the ER. Therefore, we hypothesis that PEX16 mediates the targeting of non-classic PMPs such as dPMPs by importing them first to the ER. The objectives of this research are to: 1. Determine whether dual localized proteins (dPMPs) are imported into peroxisomes by PEX16; 2. Identify the targeting signal of dPMPs for peroxisome targeting; 3. Determine the protein structure of PEX16 using cryoEM. To accomplish these objectives, we will use a combination of live-cell super-resolution microscopy with biochemical and molecular biology techniques. We will also employ the latest cryo-electron microscopy to elucidate the protein structure of PEX16. Protein targeting is essential for proper cellular function. Our proposed study is at the forefront of peroxisome protein targeting research and will provide detailed insights into how this understudied organelle recruits their proteins. Moreover, the knowledge gained from our NSERC funded research can be applied by the wider biological community to bring about broader understanding of the peroxisomes and their roles in a vast array of physiological processes.