Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D
Creators: name: Macke, Amanda J.
name: Divita, Taylor E.
name: Pachikov, Artem N.
name: Mahalingam, Sundararajan
name: Bellamkonda, Ramesh
name: Rasineni, Karuna
name: Casey, Carol A.
name: Petrosyan, Armen
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Infectious Diseases
subject: FOS: Health sciences

description: The development of alcohol-associated liver disease (ALD) is associated with disorganized Golgi apparatus and accelerated phagophore formation. While Golgi membranes may contribute to phagophores, association between Golgi alterations and macroautophagy/autophagy remains unclear. GOLGA4/p230 (golgin A4), a dimeric Golgi matrix protein, participates in phagophore formation, but the underlying mechanism is elusive. Our prior research identified ethanol (EtOH)-induced Golgi scattering, disrupting intra-Golgi trafficking and depleting RAB3D GTPase from the <i>trans</i>-Golgi. Employing various techniques, we analyzed diverse cellular and animal models representing chronic and chronic/binge alcohol consumption. In <i>trans</i>-Golgi of non-treated hepatocytes, we found a triple complex formed between RAB3D, GOLGA4, and MYH10/NMIIB (myosin, heavy polypeptide 10, non-muscle). However, EtOH-induced RAB3D downregulation led to MYH10 segregation from the Golgi, accompanied by Golgi fragmentation and tethering of the MYH10 isoform, MYH9/NMIIA, to dispersed Golgi membranes. EtOH-activated autophagic flux is evident through increased WIPI2 recruitment to the Golgi, phagophore formation, enhanced LC3B lipidation, and reduced SQSTM1/p62. Although GOLGA4 dimerization and intra-Golgi localization are unaffected, loss of RAB3D leads to an extension of the cytoplasmic N terminal domain of GOLGA4, forming GOLGA4-positive phagophores. Autophagy inhibition by hydroxychloroquine (HCQ) prevents alcohol-mediated Golgi disorganization, restores distribution of ASGR (asialoglycoprotein receptor), and mitigates COL (collagen) deposition and steatosis. In contrast to short-term exposure to HCQ, extended co-treatment with both EtOH and HCQ results in the depletion of LC3B protein via proteasomal degradation. Thus, (a) RAB3D deficiency and GOLGA4 conformational changes are pivotal in MYH9-driven, EtOH-mediated Golgiphagy, and (b) HCQ treatment holds promise as a therapeutic approach for alcohol-induced liver injury. <b>Abbreviation</b>: ACTB: actin, beta; ALD: alcohol-associated liver disease; ASGR: asialoglycoprotein receptor; AV: autophagic vacuoles; EM: electron microscopy; ER: endoplasmic reticulum; EtOH: ethanol; HCQ: hydroxychloroquine; IP: immunoprecipitation; KD: knockdown; KO: knockout; MYH10/NMIIB: myosin, heavy polypeptide 10, non-muscle; MYH9/NMIIA: myosin, heavy polypeptide 9, non-muscle; PLA: proximity ligation assay; ORO: Oil Red O staining; PM: plasma membrane; TGN: <i>trans</i>-Golgi network; SIM: structured illumination super-resolution microscopy.