ABSTRACT Methionine adenosyltransferase (MAT) is an essential cellular enzyme that catalyzes the formation of S- adenosylmethionine (SAMe), the principal biological methyl donor and in liver, precursor of the key antioxidant glutathione (GSH). In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, α1 and α2, respectively. MAT1A is primarily expressed in normal liver. Majority of patients with chronic liver disease have decreased expression and activity of MAT1A-encoded isoenzymes. We found MAT1A expression and hepatic SAMe levels are reduced in alcoholic hepatitis patients. While MAT isoenzymes are widely acknowledged for catalyzing cytosolic SAMe biosynthesis, our recent works have uncovered highly novel aspects of their functions. In addition to cytosol and nucleus, our preliminary data indicate MATα1 is also present in the mitochondrial matrix to regulate mitochondrial function. This is important as we found hepatocytes lack the mitochondrial SAMe transporter SLC25A26. Using immunoprecipitation (IP) followed by mass spectrometry, we have identified many mitochondrial proteins and cytochrome P450 2E1 (CYP2E1) as MATα1-interacting proteins. Our preliminary data show that MATα1 negatively regulates CYP2E1 expression mainly at the protein level via methylation, which has not been reported. Importantly, MATα1 mitochondrial targeting is impaired in murine and human alcoholic liver disease (ALD) and we propose two novel mechanisms that may cooperate in causing this impairment. The current proposal tests the central hypothesis that MATα1 provides the SAMe source within the hepatocyte's mitochondrial matrix and impairment in MATα1 mitochondrial targeting in ALD plays a key role in the pathogenesis of ALD. The corollary hypothesis is that MATα1 maintains hepatocyte mitochondrial function in part by suppressing CYP2E1 expression. Here we follow up these novel findings in three specific aims: 1) examine how MATα1 targets the mitochondria and why its targeting is impaired in ALD. We will elucidate how MATα1 targets the mitochondria and mechanisms of its impaired targeting in ALD. We will test the novel hypothesis that this is due to 1) increased MATα1 sumoylation and 2) increased interaction of MATα1 with PIN1 (a peptidyl-prolyl cis- trans isomerase that recognizes a specific phosphorylated motif), 2) examine how MATα1 regulates CYP2E1 protein expression. We will examine how MATα1 regulates CYP2E1 protein stability at the molecular level and identify CYP2E1 residues and interacting proteins that participate in this process, 3) examine the role of mitochondrial MATα1 and MATα1-regulated CYP2E1 in ALD. We will test the novel hypothesis that preventing MATα1 sumoylation or interaction with PIN1 will protect against reduced mitochondrial MATα1 content and injury in ALD. We will also examine whether MATα1 will protect against ALD by methylating CYP2E1 at R379 to enhance its degradation. If successfully accomplished, ...