Mechanism of targeting of Peroxisome-Mitochondria localizing proteins

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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 In our unpublished work, we have found that PEX16 can take on multiple topologies on the ER including two-transmembrane and four-transmembrane proteins.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.