Investigating the role of autophagy inducers and peroxisomal proteins in pexophagy

Investigating the role of autophagy inducers and peroxisomal proteins in pexophagy Peroxisomes are critical organelles that house fatty acid ꞵ-oxidation and antioxidative enzymes that detoxify reactive oxygen and nitrogen species. Peroxins maintain peroxisomal proteostasis by coordinating biogenesis, matrix protein import, and receptor recycling. Impairment of peroxisome biogenesis can impact many physiological processes in plants and humans. Despite the importance of peroxisomes, peroxisomal turnover, a key aspect of proteostasis, remains poorly understood. The primary mechanism for organelle turnover is autophagy, and the selective autophagy of peroxisomes is termed pexophagy. In this process, damaged or excess peroxisomes are sequestered via encirclement of an isolation membrane to form autophagosomes, which then fuse with a degradative organelle (the vacuole in plants and yeast). In the vacuole, the peroxisome, now in the autophagic body, is lysed by vacuolar proteases and lipases, and the nutrients from this degradation are exported to the cytosol for reuse by the cell. Stress-inducing conditions, such as nutrient starvation, oxidative stress, etc. can induce general autophagy; however, plant-specific pexophagy proteins and conditions remain to be identified. To better understand plant pexophagy and the proteins involved, I plan to monitor autophagy and pexophagy via western analysis of reporters that separately mark peroxisomal and autophagosomal membranes and are cleaved upon delivery of autophagosomes to the vacuole. I will also use the fluorescence of these reporters to visualize pexophagy using confocal microscopy. Using the tools that I develop, I will elucidate the role various peroxisomal proteins in pexophagy, including NBR1 (a selective autophagy receptor), peroxisomal proteins containing ATG8-interacting motifs, LON2 (a peroxisomal chaperone and protease), and peroxisome- associated ubiquitination enzymes. Beyond improving basic understanding of fundamental cell biology, furthering our understanding of peroxisome regulation can improve our understanding of metabolic illnesses in humans and provide targets for metabolic engineering in crops.