The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3 , encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change. Significance Statement Fatty liver disease affects nearly a quarter of the world’s population and has both environmental and genetic risk factors. A mutation in the gene PNPLA3 that converts Ile 148 to Met is the strongest known genetic risk factor for developing fatty liver disease. Using a series of techniques to track endogenous PNPLA3 and PNPLA3-I148M biogenesis and localization, we reveal new insights into how the mutation changes cellular dynamics. Although previous reports focus on its role on lipid droplets, we reveal that PNPLA3-I148M also functions at the Golgi apparatus, an organelle critical for protein transport into and out of the cell and lipid signaling. PNPLA3-I148M causes altered Golgi morphology and drives changes reminiscent of liver disease.