The mechanism of mammalian pexophagy is poorly understood. Although ubiquitination is thought to be involved in the process, the upstream and downstream effectors of ubiquitination remain elusive. Specifically, the E3 ligase responsible for mediating ubiquitination is not known, the site(s) of ubiquitination on the peroxisomal membrane are not known, and the key autophagy receptor which binds ubiquitin at the surface of the peroxisome is not known. In the first chapter of this thesis, I introduce the peroxisome and the key players in general autophagy, as well as in the selective autophagy of the peroxisome. In the second chapter, I explain the reagents and techniques used in this thesis. In the third chapter, I identify and characterize the peroxisomal E3 ubiquitin ligase PEX2 as the causative agent for mammalian pexophagy, identify two peroxisomal membrane proteins which are ubiquitinated by PEX2, and identify NBR1 as the primary receptor in PEX2-mediated pexophagy. In the fourth chapter, I examine the regulation of PEX2 and demonstrate how a key metabolic stimulus, protein restriction, initiates signaling through the mTORC1 pathway which leads to pexophagy in cultured cells as well as in a mouse model. Finally, I conclude my thesis with a discussion on my findings and some general thoughts on the current state of the field.
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