The N-degron pathway is a catabolic mechanism through which a wide range of cellular components are selectively degraded via the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (ALS). N-terminal arginylation, mediated by the R-transferase ATE1, destabilizes protein substrates by rapidly directing the resulting Arg/N-degrons to ubiquitination and proteasomal degradation. In the ALS, the Arg/N-degron plays a role in selective autophagy by facilitating the delivery and sequestration of various types of cargo. The autophagic N-degron pathway has been implicated in maintaining the homeostasis of subcellular organelles, including the ER, mitochondria, peroxisomes, and lipid droplets; however, its homeostatic role and molecular mechanism in innate immunity remain to be elucidated. In Chapter 1 of this dissertation, I provide a general introduction to cellular catabolic systems and the N-degron pathway. Two major systems, the UPS and ALS, work together to maintain cellular homeostasis and closely interact to effectively manage cellular stress and damage. The N-degron pathway utilizes two types of N-recognins, E3 ligases and autophagy receptors, which selectively recognize N-terminal residues to degrade N-degrons. The autophagic N-recognin p62/SQSTM1 primarily delivers Arg/N-degrons to autophagosomal membranes, while the Arg/N-degron-bearing p62 concurrently promotes autophagosome biogenesis, thereby enhancing autophagic degradation under stress conditions. In Chapter 2, I describe the role of the N-degron pathway in innate immunity and present the molecular mechanisms underlying the activation and resolution of innate immune responses in a sterile immunity model induced by proteasomal dysfunction. I show that proteasome inhibition induces the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs) via the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Cytosolic mitochondrial DNA (mtDNA), which is released in response to proteasome inhibition, triggers the immune response and is subsequently degraded through the autophagic N-degron pathway. Independent of immune response activation, the mtDNA degradation pathway is initiated by DNA-dependent protein kinase (DNA-PK), whose DNA-binding subunit, KU70, interacts with ATE1. DNA-PK-mediated activation of ATE1 induces N-terminal arginylation of BiP/GRP78 after its translocation from the endoplasmic reticulum (ER) to the cytosol. Cytosolic arginylated BiP (R-BiP), acting as a trans-Arg/N-degron, delivers cytosolic mtDNA to autophagosomes to facilitate its lysosomal degradation. Consequently, the autophagic N-degron pathway establishes a negative feedback loop that regulates cytosolic levels of mtDNA, thereby promoting the resolution of innate immune responses and preventing excessive immunity. My results demonstrate the molecular mechanisms of innate immunity under proteotoxic stress and suggest a physiological role of the N-degron pathway in the context of sterile immune responses. In Chapter 3, I elucidate the molecular mechanisms underlying the arginylation of BiP, based on the finding that cytosolic DNA is sufficient to induce BiP arginylation by ATE1. I show that BiP translocates from the ER to the cytosol in response to DNA transfection, before undergoing arginylation. This translocation is mediated by BAK, which is capable of forming membrane pores. To further investigate the regulatory mechanism of ATE1 activity, I established a BiP arginylation assay to assess the ATE1-dependent arginylation by monitoring R-BiP levels. My results identify three evolutionarily conserved residues that are critical for ATE1 enzymatic activity, and demonstrate that ATE1 isoforms, including 1A7A and 1A7B, are responsible for the BiP arginylation. For BiP recognition, the KGL motif in exon 1A and the N-terminal region in exon 1B exhibit distinct regulatory roles, suggesting that exon 1 may determine the substrate specificity of ATE1. Together, these findings reveal the molecular mechanisms underlying BiP arginylation in response to cytosolic DNA and identify key motifs in ATE1 responsible for its enzymatic activity and substrate recognition. In Chapters 4 and 5, I summarize and discuss the results of this dissertation and provide detailed descriptions of the experimental design, protocols, and materials. Although some missing links and questions remain to be addressed in future studies, the findings presented in this dissertation highlight the pathophysiological importance of the N-degron pathway in innate immunity and suggest that it could serve as a potential therapeutic target for chronic inflammation, autoimmune diseases, aging, and aging-related diseases. Furthermore, molecular elucidation of BiP arginylation paves the way for understanding the regulatory mechanism of ATE1 and protein arginylation, thereby expanding the known physiological significance of arginylation and facilitating the development of tools to modulate ATE1 activity.