Genetic Determinants of Protein Targeting from the Endoplasmic Reticulum to Lipid Droplets

Abstract

Lipid droplets (LDs) are cellular organelles specialized in storing triacylglycerol (TG). Aberrant LD accumulation is the basis of metabolic diseases like obesity and non-alcoholic fatty liver disease (NAFLD) and is associated with type 2 diabetes and cancer. Proteins on the LD surfaces facilitate organellar functions in energy homeostasis. For instance, glycero-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the rate-limiting step of the TG synthesis pathway, targets from the endoplasmic reticulum (ER) to LDs and catalyzes TG synthesis on LDs. However, how proteins target LDs remains a fundamental unsolved problem in cell biology.

To investigate the mechanism of protein targeting from the ER to LDs, we performed a genome-scale RNAi imaging screen in Drosophila S2 R+ cells to identify genetic determinants of the targeting process, using GPAT4 as a model. We found that membrane fusion machinery—including Rab1 and its activating complex component (Trs20), a membrane tethering complex component (Rint1), and four SNAREs (Syx5, membrin, Bet1, and Ykt6)—is required for LD targeting of GPAT4 and other ER proteins but not of cytosolic proteins. Based on this finding, we propose a novel model of ER-to-LD protein targeting, in which the outer leaflet of the ER bilayer membrane is fused with the LD monolayer membrane by these components to establish membrane connections that mediate protein targeting. Electron microscopy of Rab1- or Syx5-depleted cells revealed ultrastructural changes consistent with this model. Finally, we provide evidence that there exist two types of ER-LD membrane connections, only one of which mediates GPAT4 targeting to LDs.

In the screen, knockdown of genes involved in phospholipid metabolism increased GPAT4 targeting to LDs. Knockdown of TMEM19, a previously uncharacterized gene associated with human cleft malformation, also increased GPAT4 targeting to LDs. We provide bioinformatic and experimental evidence that TMEM19 is a novel enzyme involved in phospholipid metabolism and propose its catalytic activity.

My thesis leveraged the use of advanced microscopy and imaging analysis techniques to investigate a cellular protein trafficking process. Although more work is needed to elucidate the exact mechanisms, my findings open doors to understanding the fundamental cell biological process important for lipid homeostasis and human diseases.