Diacylglycerol links lipid droplets and tubular ER during growth resumption from stationary phase
Authors: Ganesan Suriakarthiga , Ponce Maria Laura Sosa , Shabits Brittney N. , Tavassoli Marjan , Zaremberg Vanina 
Published: June 2021

Diacylglycerol (DAG) is a key signaling lipid and intermediate in lipid metabolism. Stress and nutritional conditions that induce DAG synthesis also promote triacylglycerol (TAG) production and biogenesis of lipid droplets (LDs) in budding yeast. Compared to our knowledge on the spatiotemporal details leading to DAG channeling into TAG synthesis and LD emergence, we know significantly less on how DAG produced from TAG lipolysis is directed towards synthesis of phospholipids for membrane proliferation. Upon re‐entry of growth from stationary phase TAG lipolysis is mainly supported by LD‐resident lipases, providing fatty acids and DAG precursors for membrane lipid synthesis to boost rapid growth. How the metabolic flux of DAG is channeled into phospholipid synthesis while its conversion back to TAG is prevented is not well understood. It has been suggested that turnover of LDs requires a functional interaction with the ER.We monitored DAG localization in live yeast during growth resumption from stationary phase using a fluorescent DAG sensor (C1δ‐GFP). Remarkably, in the first hours after growth restarted, a diverse array of DAG‐enriched liquid ordered microdomains associated with the vacuolar membrane were observed. The formation of these domains was dependent on LD‐resident lipases Tgl3, Tgl4 and the vacuolar lipase Atg15. DAG puncta appeared adjacent to LDs positioned at vertex rings formed around apposed vacuolar membranes. Few of these DAG rich structures were found tightly associated with purified LDs obtained one hour after growth resumption. During LD purification, we identified a subphase lying beneath the LD floating layer, highly enriched in C1δ‐GFP. To gain insight into the nature of this fraction, we performed a detergent‐free immunoisolation approach to obtain membranes enriched in the DAG sensor using GFP‐Trap technology followed by proteomic analysis. A significant enrichment of proteins localized to the endoplasmic reticulum (ER) was identified. Notably, a set of structural proteins critical for the formation of tubular ER were enriched in this group. Additionally, the KEGG metabolic categories of glycerophospholipid, sphingolipid, and sterol metabolism were significantly enriched. Combined approaches using yeast genetics and pharmacological tools to study the effect of altering lipid homeostasis in all three of these categories to study their impact on DAG distribution were used. Our results suggest that DAG produced from TAG catabolism during growth resumption from stationary phase links LD consumption with the tubular ER, where a hub of lipid metabolic enzymes representing all three eukaryotic classes converge.Support or Funding InformationThis work was funded by the Natural Sciences and Engineering Research Council of Canada to VZ and by an International Eyes High Doctoral and Dean's Doctoral Award from the University of Calgary to SG and Queen Elizabeth graduate awards to MLSP and BNS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.