Posttranslational modifications of Ancient Ubiquitous Protein 1 control intracellular Lipid Droplet clustering

Abstract

Lipid droplets (LDs) are the intracellular storage organelles for neutral lipids. Over the last decade LDs have been identified to play important roles in lipid homeostasis, cellular signaling events and as a platform to sequester specific proteins. Lipid droplets are also linked to human diseases like type 2 diabetes or obesity. LDs are dynamic organelles, which can move rapidly within cells along microtubule tracks using the cellular transport machinery. While LDs are usually found evenly dispersed throughout the cytoplasm of cells, they have also been observed to aggregate and form dense clusters consisting of numerous individual LDs. It has been suggested that LD clustering is involved in LD fusion or in mediating increased storage of neutral lipids. Even though several proteins have been shown to induce LD clustering, a molecular mechanism explaining how LDs are tethered to form these clusters is unknown. <br /> This thesis demonstrates that LD associated ancient ubiquitous protein 1 (AUP1) promotes the clustering of LDs. Furthermore, it is shown that knock-down of AUP1 leads to the dispersion of LD clusters. Results from this study demonstrate that AUP1 is ubiquitinated in a process that depends on the integrity of an intrinsic AUP1 ubiquitin-binding domain. It is also shown that AUP1 is ubiquitinated at several lysine residues. AUP1 mutants that fail to become ubiquitinated lose the ability to promote LD clustering. Fusing a single ubiquitin moiety to AUP1 mutants that otherwise fail to induce LD clustering, is sufficient to restore LD clustering in cells. Thus, for the first time a posttranslational modification is identified that controls the clustering of LDs. In addition, AUP1 is known to be a highly phosphorylated protein and this study shows that mimicking phosphorylation of one specific threonine residue attenuates AUP1-induced LD clustering. It further shows that this phosphorylation of AUP1 does not interfere with the ubiquitination of AUP1. <br /> Based on these findings, a regulatory mechanism is proposed in which the identified posttranslational modifications of AUP1 control intracellular LD clustering.