PROJECT SUMMARY/ ABSTRACT Peroxisomes are ubiquitous organelles that are integrated into essential metabolic functions of eukaryotic cells such as purine catabolism, bile acid and ether phospholipid synthesis, as well as β- and α- oxidation of very long chain fatty acids (VLCFA). Deficiencies in peroxisomes have been associated with a variety of disease states, including inherited neuropathologies, aging, heart disease, cancer, and diabetes. Their importance is further underscored by the occurrence of peroxisome biogenesis disorders (PBD); serious early childhood pathologies that are often fatal and characterized by altered lipid metabolism. Peroxisomes abundance can be modulated by cellular metabolic demand via de novo synthesis at the Endoplasmic Reticulum (ER). Conversely, when they are no longer needed, peroxisomes are degraded via a selective form of autophagy known as pexophagy. Recent studies have found that peroxisomes and lipid droplets (LDs, lipid rich organelles that regulate the storage and hydrolysis of neutral lipids such as TAG and sterol esters), arise from the same ER sub-domains. The Ubiquitin-X domain 8 (UBXD8) is an ER-embedded adaptor to the p97 AAA-ATPase. At the ER UBXD8 has essential functions in ER-associated degradation (ERAD) as well as fatty acid and sterol homeostasis. Work from several groups, including our own unpublished studies indicate that UBXD8 regulates the abundance of LDs. In quantitative proteomic studies comparing the proteomes of wildtype and UBXD8 null cells, we find that loss of UBXD8 decreases the abundance of numerous peroxisomal proteins. Furthermore, from lipidomics analysis we identified an increase in VLCFAs and a decrease in cholesterol in UBXD8 knockout (KO) compared to wildtype (WT) cells. Interestingly, it is observed PBD patients accumulate VLCFAs and have consistently reduced cholesterol plasma levels. I have identified a significant decrease in peroxisome number and an increase in peroxisome size in UBXD8 KO cells relative to wildtype cells. We were further able to rescue this aberrant peroxisome phenotype by complementing UBXD8 KO cells with wildtype UBXD8. Additionally, consistent with our proteomics analyses, we found that loss of UBXD8 in different cells results in significantly lower levels of several peroxisomal proteins. Although the mechanism by which UBXD8 regulates ERAD is well understood, its role in peroxisome function is completely unknown. The proposed work will test the hypothesis that UBXD8 plays a critical role in peroxisome biogenesis at the ER. Proposed experiments will use advanced microscopy and proteomics techniques to ascertain the role of UBXD8 in peroxisome homeostasis and examine the effects of UBXD8 KO in a metabolically relevant cell line. A molecular understanding of the mechanisms and signaling pathways controlling peroxisome abundance may allow for modulation of peroxisome function during disease states.