Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Abstract

Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER).Relatively little is known about how sites of LD formation are selected, and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG synthases and additional LD biogeneiss factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG.We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.10-, 30-60-, 90-, 120-min ( Fig S4F), at 10-min, 2 h, 4 h, 21 h (Fig. 1C), and at 21 h (Fig. 1D, 4D, S1G). Cells were lysed using a Precellys 24 homogenizier, lipids were extracted as described and equal aliquots were brought to dryness. To quantify TAG and SE, lipids were spotted onto silica gel 60 thin layer chromatography (TLC) plates (Merck, Darmstadt, Germany) and developed with petroleum ether/diethylether/acetic acid (70:30:2; per vol).Glycerophospholipids were separated by TLC as described (Vaden et al., 2005). TLC plates were exposed to a tritium-sensitive screen, visualized and quantified using a phosphorimager (GE Healthcare). Brightness and contrast of the TIFF images were adjusted using Image J (NIH). Western blot analysisCells were grown over night in SC raffinose media, diluted to 1 OD600 ml in SC galactose media and cells corresponding to 3 OD600 units were harvested at 0-, 2-, 4-, 6-, 21-h time points.Samples were extracted by NaOH, and proteins were precipitated using trichloroacetic acid (TCA), and resuspended into sample buffer (Horvath and Riezman, 1994). Proteins were separated using SDS-PAGE and detected using a monoclonal antibody against GFP (Roche Diagnostics, Rotkreuz, Switzerland, dilution 1:2000), or Kar2 (Santa Cruz Biotechnology, Dallas, TX, dilution 1:5000) and using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, #2302, dilution 1:10000). Western blot experiments were performed at least twice with similar results. Quantification and statistical analysisUnless otherwise stated, data were obtained from at least three independent experiments.The images presented are representative of the results obtained. Quantitative results are expressed

Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex

“…These foci did not obviously co‐localize with the LD biogenesis factor Sei1 or LDs stained with monodansylpentane (Fig EV4D). It therefore remains unclear whether these Ice2 foci are related to Nem1‐containing sites of LD biogenesis (Adeyo et al , 2011; Choudhary et al , 2020). Strikingly, however, Spo7 and Nem1 did not form foci when Ice2 was absent (Fig 7F).…”

Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure

“…To this extent, the unexpected observation that seipin also accumulates DG, and that it does so even more efficiently than TG, is extremely intriguing. Unlike TG, DG is a constitutive ER lipid that is generally present at non negligible concentrations and that it has been shown to promote the recruitment of several LD-associated proteins, including perilipins(50), FIT proteins(51), and the TG-synthesizing acyltransferase Lro1 (31). Notably, our results are in agreement with the recent observation that seipinpositive ER sites are also enriched in DG, even in the absence of LD formation (31), and they suggest that seipin could be directly responsible for the observed DG enrichment via direct accumulation of DG molecules in its structure.…”

“…We thus investigated whether lipids other than TG could accumulate around seipin. In particular, we focused on the precursors of TG along the Kennedy pathway, DG and phosphatidic acid (PA), as both have been shown to accumulate at LD formation sites in various physiological (31) or non-physiological conditions (29). Analysis of the local enrichment factor around seipin shows that DG molecules significantly accumulate inside the seipin ring ( Figure 4A), while PA does not ( Figure S6).…”

“…The role of seipin in LD formation is potentially coupled to its function in regulating lipid metabolism (25)(26)(27), and notably that of phosphatidic acid (PA) (28)(29)(30). Recently, seipin-positive ER loci have been shown to be part of a larger protein machinery that also includes membrane and lipid remodeling proteins of the TG synthesis pathway (31), most notably, Lipin (Pah1 in yeast) and FIT proteins (Yft2 and Scs3 in . CC-BY-NC-ND 4.0 International license perpetuity.…”

Membrane shaping proteins, lipids, and cytoskeleton: Recipe for nascent lipid droplet formation

“…[ 1 ] Nem1 along with seipin establishes the LD forming sites in yeasts suggesting enzymes for neutral lipid synthesis are at LD formation sites. [ 20 ] Yeast cells lacking all four genes (ARE1, ARE2, DGA1, and LRO1) lack LDs but are still viable, pointing toward a crucial fact that neutral lipid (TAG and SE) synthesis is essential to LD formation but not for survival. In fact, TAG or SE biosynthesis is the only known “essential” factor for LD formation identified so far.…”