Peroxisome quality control: investigations of the roles of autophagy and the peroxisomal protease LON2
Young, Pierce Gregory
Doctor of Philosophy
Peroxisomes are organelles that house numerous essential reactions producing reactive oxygen or nitrogen species as byproducts. Peroxisomes derive their name from hydrogen peroxide, which peroxisomes can detoxify using the enzyme catalase, but the hydrogen peroxide along with other reactive oxygen and nitrogen species can damage the resident peroxisomal enzymes. Cells employ multiple mechanisms to maintain healthy populations of peroxisomes. In one peroxisome quality control mechanism, entire damaged or superfluous peroxisomes are degraded through autophagy, which is known as pexophagy when peroxisomes are selectively targeted. However, how healthy and damaged peroxisomes are distinguished is unknown. In another peroxisome quality control mechanism, the peroxisomal protease LON2 in plant cells is positioned to degrade or refold damaged proteins inside peroxisomes, but much about the function of LON2 is unknown, including its substrates. To gain a better understanding of autophagy and LON2-mediated peroxisomal homeostasis, I took a variety of approaches using the reference plant Arabidopsis thaliana. I conducted a forward-genetic screen for lon2 suppressors. Arabidopsis lon2 mutants exhibit heightened pexophagy, resulting in several easily assayable phenotypes. I recovered 26 lon2 suppressors disrupting several AUTOPHAGY-RELATED (ATG) genes: ATG2, ATG3, ATG5, ATG7, ATG16, and ATG18a. Three of these genes lack T-DNA insertional alleles in publicly available repositories, and the mutants that I recovered will be useful for future studies investing the functions of these genes. I also demonstrated that an insertional atg11 allele only partially suppresses lon2 defects and that the selective autophagy receptor NBR1 is neither necessary for lon2 pexophagy nor sufficient for pexophagy in wild-type seedlings, indicating that Arabidopsis can utilize an NBR1-independent mechanism to target peroxisomes for degradation. I also characterized a series of lon2 alleles and used site-directed mutagenesis to generate multiple lon2 protein variants that will be useful in interrogating the functions of LON2. Finally, I developed a variety of fluorescent peroxisomal membrane reporters that will be useful for investigating pexophagy and other aspects of peroxisome biology in Arabidopsis. My research highlights the interconnection of autophagy and LON2 in peroxisome quality control and lays the groundwork for future studies elucidating this fascinating process.
Peroxisome; LON2; pexophagy; autophagy