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1Department of Biochemistry and Molecular Biology, College of Medicine, and 2Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA, 3Omaha Western Iowa Health Care System, VA Service, Department of Research Service, Omaha, NE, USA

The abnormalities in the Golgi apparatus function are important for the development of alcoholic liver injury, but mechanism and consequences have not been defined. Previously, we found that formation of compact Golgi requires dimerization of the largest Golgi matrix protein, giantin, which is catalyzed by protein disulfide isomerase A3 (PDIA3). Here, in both HepG2 cells expressing alcohol dehydrogenase and hepatocytes isolated from alcohol-fed rats, we show that ethanol administration induces crucial Golgi disorganization, as reflected by conversion of its main body to the several mini-Golgi structures, exhibiting swollen and distended cisternae. This Golgi fragmentation was accompanied by reduced level of giantin and its dimer form, and surprisingly, by decreased content of Sar1a, the small GTPase which initiates formation of COPII vesicles. Further analysis revealed that ethanol blocks activation of Sar1a, thus preventing formation of COPII. We found that PDIA3 employs a COPII-dependent mechanism for Golgi targeting and that after ethanol treatment, this enzyme is arrested in the endoplasmic reticulum (ER), thus blocking formation of giantin dimer. Notably, Sar1a gene silencing in hepatocytes mimics the effect of ethanol: dedimerization of giantin, trapping PDIA3 in the ER, and large-scale alterations in Golgi morphology. EtOH induced Golgi disorganization appears to have no effect on ER-to-Golgi transportation of the hepatic asialoglycoprotein receptor (ASGP-R), but it does result in its deposition in cis-medial-, but not trans-Golgi, thereby preventing its delivery to the plasma membrane. Further, we found that EtOH administration results in S1943 phosphorylation of non-muscle Myosin IIA (NMIIA) heavy chain, thus facilitating its connection with Golgi enzymes, as detected by biochemical approaches and 3D Structured Illumination Microscopy. We revealed that NMIIA-P-S1943 competes with giantin for the Rab6a GTPase dimer, which was converted to monomer after Golgi fragmentation. Therefore, Rab6a plays a dual role in the Golgi, serving as master regulator of Golgi organization/disorganization, and NMIIA and Giantin have a ‘tag-of-war’ to modulate Golgi organization. Downregulation of NMIIA or overexpression of NMHC-IIAΔtailpiece, as well overexpression of dominant negative Rab6a(T27N), a GDP-bound mutant, preserved compact Golgi phenotype. Thus, targeting of NMIIA-P-S1943 may be important for prevention of alcohol metabolism’s damaging effects on the cell.

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