Abstract 1408 "Nuclear lipid droplet characterization in budding yeast"

Lipid droplets (LDs) are often present in the cytoplasm of eukaryotes, but in some cell types such as hepatocytes or budding yeast, a number of LDs are found in the nucleus (nLDs) in response to stress. The physiological function of nLDs needs additional studies, as well as why they are abundant in only limited cell types. The inner nuclear membrane is involved in the formation of nLDs in both mammalian hepatocytes and budding yeast. We have previously shown that alterations of the nuclear envelope morphology by a non-metabolizable lysophosphatidylcholine (LysoPC) analogue led to drastic changes in gene expression driven by Mga2, Spt23 and Opi1 in their dual role of membrane property sensors and transcriptional regulators. Expression of the only yeast Δ9-desaturase Ole1 and phospholipid remodelling pathways were upregulated while all branches of the de novo phospholipid synthesis downstream of phosphatidic acid (PA) were repressed. This rewiring of lipid metabolism in response to LysoPC accumulation resulted in nLDs formation. Little is known about the proteome and lipidome of yeast nLDs. The biogenesis of both cLDs and nLDs depends on PA. The first reaction in the de novo synthesis of PA is catalyzed by two glycerol-3 phosphate acyltransferases (GPATs) in yeast, Sct1 and Gpt2. Gpt2 can also use DHAP as substrate. In this work we show that Gpt2 (but not Sct1) contributes to nLDs generation in response to a LysoPC burden. A differential role of Gpt2 was first suggested by RNAseq data from cells treated with the LysoPC analogue. Second, nLD biogenesis induced by the LysoPC drug was dependent on Gpt2 as seen by using deletion mutant strains sct1Δ and gpt2Δ expressing DsRed-HDEL to delineate the NE and stained with Bodipy® to detect LDs. Preliminary results from a thorough analysis of known LD-resident proteins, has identified Opi1-GFP association with nLDs. A strategy to enhance the accumulation of nLDs by treatment of yeast GPAT mutants with the LysoPC analogue combined with Opi1 pulldown is currently being tested to aid in nLD purification for proteomic and lipidomic future characterization. This work has been financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant to VZ