UC Berkeley

The endoplasmic reticulum (ER) serves as the entry point to the secretory system where nearly one-third of the cellular proteome must undergo synthesis, folding, and maturation events before being deployed. Proteins that fail to successfully navigate these processes and achieve their native conformation are detained by endoplasmic reticulum-associated degradation (ERAD), a quality-control mechanism responsible for targeting misfolded proteins for degradation by the cytosolic 26S proteasome4. Recent studies have demonstrated that treatment with the long chain acyl-CoA synthetase inhibitor triacsin c disrupts lipid droplet (LD) biogenesis and ERAD, suggesting a functional connection between the processes. However, whether LDs are involved in ERAD remains an outstanding question.

LDs are highly dynamic neutral lipid storage organelles that function as central hubs of lipid metabolism charged with storing lipids and maintaining energy homeostasis of the cell. The specific metabolic role of LDs is dictated by the cell type and the metabolic state of the cell, which can fluctuate in response to a number of cellular stimuli. LD functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. The ability to define a high-confidence LD proteome is paramount to understanding LD functions and dynamics. However, accurate analysis of the LD proteome composition has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions.

In chapter one, we discuss the connection between protein and lipid regulatory systems within the ER and LDs, highlighting the importance of ERAD and lipophagy in maintaining cellular homeostasis. In chapter two, we use chemical and genetic approaches to disrupt LD biogenesis to explore a potential role for LDs in ERAD, ultimately providing evidence that LDs are dispensable for mammalian ERAD. Instead, our results suggest that triacsin c causes global alterations to the lipid landscape that disrupt ER proteostasis by interfering with the glycan trimming and dislocation steps of ERAD. Finally, in chapter three we develop a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. We apply this approach to two different cell types and are able to identify the vast majority of previously validated LD proteins, exclude common contaminating proteins, and identify the autophagy adaptor p62 as a mediator of hepatic lipophagy. Together these studies advance our understanding of the mechanisms that regulate lipid dynamics in the ER and LDs and their contribution towards maintaining cellular homeostasis.