ABSTRACT This proposal is a competitive renewal of a multiple-PI R01 funded through NIAAA. The goal of the application is to examine how ethanol (EtOH) exposure contributes to fat accumulation in the liver due to altered dynamic properties of the lipid droplet (LD), a fat storage organelle. Almost all heavy drinkers develop fatty liver, which is marked by the aberrant and significant accumulation of intrahepatocellular triglycerides stored within LDs. Understanding the cellular processes contributing to this fat accumulation will provide essential information for preventing further injury progression, as it is known that alcoholic fatty liver is the initial but reversible stage of liver injury. Here, we propose to provide molecular insights into these critical questions, aimed at potential treatments that reverse or prevent EtOH-induced steatosis. This proposal combines the expertise of two senior investigators recognized for their contributions to the study of EtOH-induced cell injury and hepatic LD biology. We believe this collaborative effort has been beneficial to the field of ALD and will result in outcomes not attainable by individual efforts alone. This proposal comprises two well-integrated aims. Aim One: Blockage of ER-Associated Lipophagy by EtOH. We have made the interesting observation that nascent LDs of a conserved size (170nm) accumulate at the ER surface of EtOH-damaged hepatocytes and that lysosomal/autophagic compartments subsequently catabolize these ER-associated LDs. Notably, both chronic EtOH exposure and pharmacologic disruption of lysosome function attenuate this process leading to hepatocyte steatosis. Aim Two: Disruptive Alterations of the LD Proteome by EtOH. We found that many components of the LD “surface proteome” are post-translationally modified by ubiquitin, a pathway we posit directs removal of select proteins to the proteasome for degradation or directs the entire LD to the lysosome for catabolism. Importantly, we have found that chronic EtOH exposure markedly increases the ubiquitination of the LD proteome and thus disrupts normal LD degradation/catabolism leading to steatosis. These observations and specific aims will allow us to pursue the CENTRAL HYPOTHESIS of this study that EtOH promotes accumulation of both mature and nascent, ER-associated, LDs through alterations of the LD proteome inhibiting lipophagy and lipid catabolism leading to hepatocyte steatosis. The proposed investigation will utilize state-of- the-art imaging and proteomic technologies to quantify specific molecular events that contribute to alcohol- induced fatty liver. Successful completion of these studies will provide novel insights as to how EtOH affects LD dynamics in liver cells and important information for therapeutic strategies aimed at reducing or eliminating the severity of steatosis and blocking its further progression to alcoholic steatohepatitis, fibrosis, and cirrhosis.