Tubular ER Associates With Diacylglycerol-Rich Structures During Lipid Droplet Consumption.

Growth resumption from stationary phase in Saccharomyces cerevisiae, is characterized by lipid droplet (LD) consumption and channeling of lipid precursors toward synthesis of membranes. We have previously determined that triacylglycerol lipolysis contributes to a pool of diacylglycerol (DAG) associated with the yeast vacuole that is enriched in structures that are in close proximity to LDs. In this study we have monitored these structures using a DAG sensor fused to GFP during isolation of LDs. A unique fraction containing the DAG sensor, with low presence of LDs, was identified. Membranes enriched in the DAG probe were obtained by immunoaffinity purification using a GFP nanobody, and the associated proteome was investigated by mass spectrometry. It was determined this LD-associated fraction was enriched in proteins known to shape the tubular endoplasmic reticulum (ER) like Yop1, Sey1, Rtn1, and Rtn2. Consistently, cells lacking three of these proteins (rtn1Δ rtn2Δ yop1Δ) exhibited delayed LD consumption, larger LDs and abnormal LD distribution. In addition, the triple mutant displayed aberrant localization of the DAG sensor after 5 h of growth resumption from stationary phase. Manipulation of DAG levels by overexpression of the DAG kinase Dgk1, impacted localization of the DAG probe and affected fitness of the triple mutant. Altogether these results link LD consumption to tubular ER expansion as a gateway of lipid precursors that otherwise accumulate in vacuolar associated membranes or other internal compartments. Furthermore, conversion of DAG to phosphatidic acid (PA) in the absence of a functional tubular ER was toxic to cells, suggesting the ratio of PA to DAG is critical to allow growth progression.

Phosphorylation of the lipid droplet localized glycerol­3­phosphate acyltransferase Gpt2 prevents a futile triacylglycerol cycle in yeast.

The proteome of lipid droplets, storage compartments of triacylglycerols (TAGs), comprises TAG synthesizing and TAG degrading enzymes. Thus, to prevent a futile cycle the activity of enzymes catalyzing key steps in TAG turnover has to be strictly coordinated. The first and committed reaction of TAG synthesis is catalyzed by a glycerol­3­phosphate acyltransferase (GPAT). Here we demonstrate that in the model organism yeast the lipid droplet associated GPAT Gpt2 requires phosphorylation at a conserved motif to prevent a futile TAG cycle. Phosphorylation deficiency at the conserved motif increases the enzyme activity of Gpt2 and consequently enhances TAG synthesis. In proliferating cells the phosphorylation deficient GPAT-form contributes to TAG metabolism similar to control. However, during lipolysis the increased activity of phosphorylation deficient Gpt2 causes a constant TAG level by using TAG-released fatty acids as substrate for TAG synthesis. These data strongly indicate that phosphorylation of Gpt2 at a conserved motif plays a critical role in coordinating the synthesis and degradation of TAGs.

Metabolic control of cytosolic-facing pools of diacylglycerol in budding yeast.

Diacylglycerol (DAG) is a key signaling lipid and intermediate in lipid metabolism. Our knowledge of DAG distribution and dynamics in cell membranes is limited. Using live-cell fluorescence microscopy we investigated the localization of yeast cytosolic-facing pools of DAG in response to conditions where lipid homeostasis and DAG levels were known to be altered. Two main pools were monitored over time using DAG sensors. One pool was associated with vacuolar membranes and the other localized to sites of polarized growth. Dynamic changes in DAG distribution were observed during resumption of growth from stationary phase, when DAG is used to support phospholipid synthesis for membrane proliferation. Vacuolar membranes experienced constant morphological changes displaying DAG enriched microdomains coexisting with liquid-disordered areas demarcated by Vph1. Formation of these domains was dependent on triacylglycerol (TAG) lipolysis. DAG domains and puncta were closely connected to lipid droplets. Lack of conversion of DAG to phosphatidate in growth conditions dependent on TAG mobilization, led to the accumulation of DAG in a vacuolar-associated compartment, impacting the polarized distribution of DAG at budding sites. DAG polarization was also regulated by phosphatidylserine synthesis/traffic and sphingolipid synthesis in the Golgi.

Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast.

Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed.