Reprogrammed lipid metabolism protects inner nuclear membrane against unsaturated fat

•Biosensors detect lipid saturation dynamics of INM•Increased lipid unsaturation induces LDs at ONM, but not at INM•Opposing transcription circuits reprogram LD synthesis across the NE•LDs detoxify unsaturated lipids to maintain INM integrity The cell nucleus is surrounded by a double membrane. The lipid packing and viscosity of membranes is critical for their function and is tightly controlled by lipid saturation. Circuits regulating the lipid saturation of the outer nuclear membrane (ONM) and contiguous endoplasmic reticulum (ER) are known. However, how lipid saturation is controlled in the inner nuclear membrane (INM) has remained enigmatic. Using INM biosensors and targeted genetic manipulations, we show that increased lipid unsaturation causes a reprogramming of lipid storage metabolism across the nuclear envelope (NE). Cells induce lipid droplet (LD) formation specifically from the distant ONM/ER, whereas LD formation at the INM is suppressed. In doing so, unsaturated fatty acids are shifted away from the INM. We identify the transcription circuits that topologically reprogram LD synthesis and identify seipin and phosphatidic acid as critical effectors. Our study suggests a detoxification mechanism protecting the INM from excess lipid unsaturation. The cell nucleus is surrounded by a double membrane. The lipid packing and viscosity of membranes is critical for their function and is tightly controlled by lipid saturation. Circuits regulating the lipid saturation of the outer nuclear membrane (ONM) and contiguous endoplasmic reticulum (ER) are known. However, how lipid saturation is controlled in the inner nuclear membrane (INM) has remained enigmatic. Using INM biosensors and targeted genetic manipulations, we show that increased lipid unsaturation causes a reprogramming of lipid storage metabolism across the nuclear envelope (NE). Cells induce lipid droplet (LD) formation specifically from the distant ONM/ER, whereas LD formation at the INM is suppressed. In doing so, unsaturated fatty acids are shifted away from the INM. We identify the transcription circuits that topologically reprogram LD synthesis and identify seipin and phosphatidic acid as critical effectors. Our study suggests a detoxification mechanism protecting the INM from excess lipid unsaturation. The membranes of different organelles vary considerably in lipid composition and hence functionality (Bigay and Antonny, 2012Bigay J. Antonny B. Curvature, lipid packing, and electrostatics of membrane organelles: defining cellular territories in determining specificity.Dev. Cell. 2012; 23: 886-895Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar; van Meer et al., 2008van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4144) Google Scholar). The endoplasmic reticulum (ER) is a complex organelle of highly specialized subdomains comprising the nuclear envelope (NE) and the peripheral ER. The peripheral ER consists of cytoplasmic cisternae, tubules, and a plasma-membrane-associated domain in yeast (West et al., 2011West M. Zurek N. Hoenger A. Voeltz G.K. A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature.J. Cell Biol. 2011; 193: 333-346Crossref PubMed Scopus (238) Google Scholar). The outer nuclear membrane (ONM) and peripheral ER produce glycerophospholipids (or phospholipids in short; PL) for membrane growth, and triacylglycerol (TAG) to stockpile energy (Carman and Han, 2011Carman G.M. Han G.S. Regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae.Annu. Rev. Biochem. 2011; 80: 859-883Crossref PubMed Scopus (168) Google Scholar). The inner nuclear membrane (INM) is in direct contact with the genome, and its point of contact with the ONM and ER is at nuclear pore complexes (Ungricht and Kutay, 2017Ungricht R. Kutay U. Mechanisms and functions of nuclear envelope remodelling.Nat. Rev. Mol. Cell Biol. 2017; 18: 229-245Crossref PubMed Scopus (167) Google Scholar). Active lipid metabolism at the INM enables cells to store fatty acids (FAs) in nuclear lipid droplets (nLDs) (Barbosa et al., 2019Barbosa A.D. Lim K. Mari M. Edgar J.R. Gal L. Sterk P. Jenkins B.J. Koulman A. Savage D.B. Schuldiner M. et al.Compartmentalized synthesis of triacylglycerol at the inner nuclear membrane regulates nuclear organization.Dev. Cell. 2019; 50: 755-766.e6Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar; Romanauska and Köhler, 2018Romanauska A. Köhler A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets.Cell. 2018; 174: 700-715.e18Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). As a result of its lipid metabolism, the INM has a distinct lipid composition compared with the ONM featuring high levels of diacylglycerol, a precursor for both PL and TAG synthesis. How cells sense and adjust the lipid properties of the INM in various metabolic states is a key open question. Fatty acyl chains, esterified in glycerophospholipids, form the hydrophobic barrier of biological membranes and determine their viscosity, thickness, water permeability, and bending rigidity (Ernst et al., 2016Ernst R. Ejsing C.S. Antonny B. Homeoviscous adaptation and the regulation of membrane lipids.J. Mol. Biol. 2016; 428: 4776-4791Crossref PubMed Scopus (152) Google Scholar). Lipids with saturated acyl chains are packed more tightly and tend to form non-fluid gel phases; mono- and polyunsaturated acyl chains have kinked shapes, which fluidize bilayers. The collective biophysical properties of membranes also profoundly affect membrane-embedded proteins in their structure, activity, and signaling behavior. The fatty acyl chain profile of membranes reflects a balance between endogenous FA synthesis, recycling of FAs from lipid breakdown, and FA uptake from the exterior. Nothing is known about the fatty acyl chain composition of the INM and, hence, how INM lipid packing and viscosity, or its reciprocal, fluidity, are regulated. In budding yeast, Ole1 is the sole enzyme that can introduce a double bond into fatty acyl chains (Martin et al., 2007Martin C.E. Oh C.S. Jiang Y.D. Regulation of long chain unsaturated fatty acid synthesis in yeast.Biochim. Biophys. Acta. 2007; 1771: 271-285Crossref PubMed Scopus (154) Google Scholar). There is no opposing enzymatic activity making this reaction irreversible. Ole1 is located in the ER and specifically introduces a double bond at the C9 position. The expression of Ole1 is controlled by the cellular lipid acyl chain composition and is strongly reduced when unsaturated fatty acids (UFAs) are abundant (Figure 1A). This feedback control depends on the sensor proteins Mga2 and Spt23, which are produced as homo-dimeric, ER-bound, inactive precursors of transcription factors (Covino et al., 2016Covino R. Ballweg S. Stordeur C. Michaelis J.B. Puth K. Wernig F. Bahrami A. Ernst A.M. Hummer G. Ernst R. A eukaryotic sensor for membrane lipid saturation.Mol. Cell. 2016; 63: 49-59Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar; Hoppe et al., 2000Hoppe T. Matuschewski K. Rape M. Schlenker S. Ulrich H.D. Jentsch S. Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.Cell. 2000; 102: 577-586Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). The crucial element for UFA sensing is Mga2’s transmembrane helix (Figure 1A), which harbors specific sensory residues embedded deep in the lipid bilayer (Covino et al., 2016Covino R. Ballweg S. Stordeur C. Michaelis J.B. Puth K. Wernig F. Bahrami A. Ernst A.M. Hummer G. Ernst R. A eukaryotic sensor for membrane lipid saturation.Mol. Cell. 2016; 63: 49-59Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The transmembrane helices continuously explore alternative rotational states. Loose lipid packing (high UFA content) stabilizes conformations where two sensory tryptophan residues point away from the dimer interface toward the lipid environment. Tight lipid packing (low UFA content) stabilizes alternative rotational conformations with the sensory tryptophans facing each other in the dimer interface. Thus, Mga2 operates via a rotation-based mechanism: the transmembrane helices sense the membrane state and through conformational changes transduce this signal to Mga2’s ubiquitination sites (Covino et al., 2016Covino R. Ballweg S. Stordeur C. Michaelis J.B. Puth K. Wernig F. Bahrami A. Ernst A.M. Hummer G. Ernst R. A eukaryotic sensor for membrane lipid saturation.Mol. Cell. 2016; 63: 49-59Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). When unsaturated lipids are scarce and the lipid packing density is high, Mga2 and Spt23 are ubiquitinated by the E3 ligase Rsp5, partially processed by the proteasome to release the active transcription factor, and imported into the nucleus via a cryptic nuclear localization sequence (NLS) to increase Ole1 expression (Piwko and Jentsch, 2006Piwko W. Jentsch S. Proteasome-mediated protein processing by bidirectional degradation initiated from an internal site.Nat. Struct. Mol. Biol. 2006; 13: 691-697Crossref PubMed Scopus (82) Google Scholar; Rape et al., 2001Rape M. Hoppe T. Gorr I. Kalocay M. Richly H. Jentsch S. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone.Cell. 2001; 107: 667-677Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) (Figures 1A and 1B). In contrast, when unsaturated lipids are abundant and the lipid packing density is low, these transcription factors remain tethered to the ER. Nutrient-derived FAs can be toxic, leading to ER stress, proliferation of ER membranes, and finally lipoapoptosis (Garbarino et al., 2009Garbarino J. Padamsee M. Wilcox L. Oelkers P.M. D'Ambrosio D. Ruggles K.V. Ramsey N. Jabado O. Turkish A. Sturley S.L. Sterol and diacylglycerol acyltransferase deficiency triggers fatty acid-mediated cell death.J. Biol. Chem. 2009; 284: 30994-31005Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar; Petschnigg et al., 2009Petschnigg J. Wolinski H. Kolb D. Zellnig G. Kurat C.F. Natter K. Kohlwein S.D. Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast.J. Biol. Chem. 2009; 284: 30981-30993Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). UFAs seem to be particularly toxic and are mainly channeled into neutral lipids with subsequent biogenesis of LDs. LDs appear to be critical as a storage space for potentially harmful UFAs (Fakas et al., 2011Fakas S. Qiu Y.X. Dixon J.L. Han G.S. Ruggles K.V. Garbarino J. Sturley S.L. Carman G.M. Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.J. Biol. Chem. 2011; 286: 29074-29085Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar; Velázquez et al., 2016Velázquez A.P. Tatsuta T. Ghillebert R. Drescher I. Graef M. Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation.J. Cell Biol. 2016; 212: 621-631Crossref PubMed Scopus (95) Google Scholar), but the molecular mechanisms of UFA detoxification are poorly understood. A particular problem is to separate the pleiotropic effects of nutrient overload from the specific effects of acyl chain saturation. Here, we have employed a targeted genetic strategy to introduce a single double bond in FAs without nutrient overload. UFAs probably reach the INM by lateral diffusion from the ONM. If and how the INM controls its saturation status is unknown and of potentially significant physiologic consequences considering its specialized proteome and intimate contact with the genome. We previously observed that oleic acid supplementation (a monounsaturated FA) or mutations in Cds1, a key enzyme in PL synthesis, induced LD formation from both the INM and ONM, creating nuclear and cytoplasmic LDs (nLDs and cLDs), respectively (Romanauska and Köhler, 2018Romanauska A. Köhler A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets.Cell. 2018; 174: 700-715.e18Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). We therefore sought to explore whether LD formation at the NE contributes to detoxifying unsaturated acyl chains from the INM. In this study, we have developed tools to directly examine lipid (un-)saturation at the INM in living cells. We provide evidence that cells orchestrate lipid metabolism across the NE by channeling potentially harmful UFAs into cytoplasmic, but not nuclear, LDs, thus protecting the INM and the nucleus from UFA-mediated lipotoxicity. Since the INM is capable of lipid metabolism, we asked how this specialized membrane territory, adjacent to the genome, responds to changes in lipid saturation. We developed genetically encoded, fluorescently labeled lipid saturation (LipSat) sensors, which measure fatty acyl chain saturation in phospholipids specifically at the INM, or, across the entire ER/NE network. These LipSat sensors are based on S.cerevisiae Mga2 (Figure 1A) and are targeted to the INM or the ER/NE by appending or omitting, respectively, the NLS of the INM transmembrane protein Heh2 (Meinema et al., 2011Meinema A.C. Laba J.K. Hapsari R.A. Otten R. Mulder F.A.A. Kralt A. van den Bogaart G. Lusk C.P. Poolman B. Veenhoff L.M. Long unfolded linkers facilitate membrane protein import through the nuclear pore complex.Science. 2011; 333: 90-93Crossref PubMed Scopus (85) Google Scholar) (Figure 1B). The Heh2 NLS directs lateral diffusion from the ONM to the INM via the nuclear pore complex. This import mechanism is distinct from that of the processed, soluble part of Mga2, which is mediated by a cryptic NLS (Figure 1B). We deleted the transcriptional activation domain (AD) of Mga2 to uncouple UFA sensing from Ole1 transcription (Hoppe et al., 2000Hoppe T. Matuschewski K. Rape M. Schlenker S. Ulrich H.D. Jentsch S. Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.Cell. 2000; 102: 577-586Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). We expected an mGFP-labeled fragment of the LipSat sensors (p90∗) to be clipped off the membrane by the proteasome when membrane lipids are saturated (i.e., UFA levels are low) (Figures 1B–1D) and to accumulate in the nucleoplasm due to its cryptic NLS. In contrast, the LipSat sensors would remain membrane bound (p120∗) when membrane lipids are unsaturated (i.e., cellular UFA content is high) (Figures 1C and 1D). The sensors were expressed in mga2Δ cells to avoid hetero-dimerization with wild-type Mga2. They were expressed at near endogenous levels and did not affect cell growth under the conditions tested (Figures S1A and S1B). The control mga2Δ cells, which have reduced Ole1 activity (Martin et al., 2007Martin C.E. Oh C.S. Jiang Y.D. Regulation of long chain unsaturated fatty acid synthesis in yeast.Biochim. Biophys. Acta. 2007; 1771: 271-285Crossref PubMed Scopus (154) Google Scholar), exhibited a largely processed LipSat sensor, consistent with a lack of unsaturated fatty acyl chains (Figures 1E–1H). To confirm that the LipSat sensors faithfully detect the degree of fatty acyl chain saturation in membranes, we supplemented the media with saturated stearic acid (C18:0), monounsaturated oleic acid (C18:1) or di-unsaturated linoleic acid (C18:2). These FAs are metabolized and incorporated into PLs, hence altering their degree of saturation. As a quantitative readout, we determined the amount of nucleoplasmic mGFP-p90∗ versus the membrane-bound mGFP-p120∗ protein by fluorescence microscopy and by immunoblotting. The INM LipSat sensor was found mostly unprocessed at the INM when cells were supplemented with linoleic acid (C18:2) or oleic acid (C18:1) (Figures 1E–1H), as expected for increased UFA levels. By comparison, cells supplemented with fully saturated stearic acid (C18:0) showed enhanced sensor processing and accumulation of mGFP-p90∗ in the nucleoplasm (Figures 1E–1H). Hence, nutrient-derived unsaturated acyl chains can accumulate in the INM. Wild-type cells contained more unprocessed sensor at the INM than mga2Δ cells, indicating that Ole1 unsaturase activity increases UFAs at the INM, but not to the same extent as the exogenously supplied UFAs (Figures S1C–S1F). Importantly, mutating the sensory W1042 and P1044 residues in the transmembrane helix of the INM LipSat recapitulates the sensing defect that was previously reported for endogenous Mga2 (Covino et al., 2016Covino R. Ballweg S. Stordeur C. Michaelis J.B. Puth K. Wernig F. Bahrami A. Ernst A.M. Hummer G. Ernst R. A eukaryotic sensor for membrane lipid saturation.Mol. Cell. 2016; 63: 49-59Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This evidence supports the notion that the Mga2-derived biosensors use the same rotation-based UFA sensing mechanism as endogenous Mga2 (Figures 1I–1K). In parallel, we characterized the global ER LipSat sensor, which localized to both the peripheral ER and the NE. This sensor’s response to UFAs mirrored the behavior of the INM sensor: it remained unprocessed and membrane bound when UFA levels were increased (i.e., linoleic acid) but became processed and nucleoplasmic when UFA levels decreased (i.e., stearic acid) (Figures S1G–S1J). The INM LipSat sensor responded more strongly to changes in fatty acyl chain saturation levels compared with the global ER LipSat sensor (see Figures 1H and S1J). This might reflect a physiologically relevant hypersensitivity of the INM toward lipid unsaturation or a greater accumulation of unsaturated lipids in this membrane territory. In sum, our lipid saturation sensors can detect nutrient-induced fatty acyl chain profiles of the INM or the entire ER network. Budding yeast cells take up various exogenous UFAs, including UFAs they cannot produce themselves like linoleic acid (C18:2) (Martin et al., 2007Martin C.E. Oh C.S. Jiang Y.D. Regulation of long chain unsaturated fatty acid synthesis in yeast.Biochim. Biophys. Acta. 2007; 1771: 271-285Crossref PubMed Scopus (154) Google Scholar). The broad substrate specificity of acyl-CoA synthetases may explain the ability of yeast to incorporate non-native, polyunsaturated FAs from their environment (Black and DiRusso, 2007Black P.N. DiRusso C.C. Yeast acyl-CoA synthetases at the crossroads of fatty acid metabolism and regulation.Biochimica et Biophysica Acta (BBA) - Molecular. and. Cell. Biology. of. Lipids. 2007; 1771: 286-298Crossref PubMed Scopus (148) Google Scholar). In contrast, Ole1 (human SCD1), the sole fatty acid desaturase in yeast, only introduces a single double bond at the C9 position of palmitic (C16:0) and stearic (C18:0) acid. To test whether the LipSat sensors detect this endogenous unsaturase activity, we expressed Ole1 from a strong GPD promoter in mga2Δ cells (Figure 2A). This resulted in a largely unprocessed, INM-bound LipSat sensor demonstrating that Ole1 increases PL unsaturation of the INM (see Figures 2A and 2B for sensor localization, and Figures 2C and 2D for quantitative immunoblotting). In contrast, the LipSat sensor was processed in mga2Δ control cells containing an empty vector, indicating high INM saturation levels (Figures 2A–2D). A similar sensor response was seen for the global ER LipSat sensor (Figures S2A–S2D). Thus, Ole1 activity directly affects the INM and ER/ONM acyl chain profile. To verify that the INM LipSat sensor measures lipid saturation of the INM rather than the ER/ONM, we confirmed that sensing and proteasomal processing (Figure 1A) indeed take place at the INM. We generated LipSat sensors tagged with mGFP and mCherry at the N and C terminus, respectively (Figure S1K), and overexpressed Ole1 to increase cellular UFA content. If the sensor was processed at the ER, the mCherry-containing transmembrane helix should remain in the ER, whereas the mGFP-p90∗ fragment is targeted to the nucleoplasm (Figures S1L and S1M). Importantly, the mCherry-transmembrane helix of the INM LipSat sensor was not detected at the peripheral ER, consistent with the idea that UFA sensing and sensor processing take place directly at the INM (Figure S1N). In contrast, the mCherry-transmembrane helix of the ER LipSat sensor was detected at the ER (Figure S1N). Finally, we ascertained that the LipSat sensors are inert, such that sensing at the ONM/ER does not influence UFA accumulation at the INM and vice versa. The INM LipSat sensor, co-expressed with an ER LipSat sensor, showed a similar UFA response when compared with cells that expressed the INM LipSat or ER LipSat sensor alone. Hence, the two sensors, expressed in different membrane compartments do not interfere with each other (Figures S2E–S2G). Taken together, the development of compartment-specific LipSat biosensors enabled us to show that the INM experiences changes in PL saturation that are induced by exogenous UFAs or Ole1 activity. Upon FA overload, cells produce excess TAG, which is stored in LDs (Henne et al., 2020Henne M. Goodman J.M. Hariri H. Spatial compartmentalization of lipid droplet biogenesis.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2020; 1865: 158499Crossref PubMed Scopus (25) Google Scholar; Olzmann and Carvalho, 2019Olzmann J.A. Carvalho P. Dynamics and functions of lipid droplets.Nat. Rev. Mol. Cell Biol. 2019; 20: 137-155Crossref PubMed Scopus (562) Google Scholar; Walther et al., 2017Walther T.C. Chung J. Farese R.V. Lipid droplet biogenesis.Annu. Rev. Cell Dev. Biol. 2017; 33: 491-510Crossref PubMed Scopus (290) Google Scholar). In principle, LDs can be synthesized either from the ONM/peripheral ER (cLDs), or from the INM (nLDs). The decision-making behind the site of LD formation (cytoplasm versus nucleoplasm) is unknown; however, high INM phosphatidic acid (PA) levels may favor nLD production (Romanauska and Köhler, 2018Romanauska A. Köhler A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets.Cell. 2018; 174: 700-715.e18Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). To find out how the saturation status of FAs affects INM lipid metabolism and nLD formation, we supplemented wild-type cells with equimolar amounts of SFAs (stearic acid, C18:0) and UFAs (oleic acid, C18:1 or linoleic acid, C18:2) (Figure 2E). To detect nLDs, we employed a previously established PA biosensor (NLS-Opi1 Q2-mCherry) in conjunction with the BODIPY 493/503 dye that stains neutral lipids. During nLD synthesis, this NLS-PA sensor becomes first enriched at the INM and subsequently on nLDs, which are connected to the INM by membrane bridges (Romanauska and Köhler, 2018Romanauska A. Köhler A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets.Cell. 2018; 174: 700-715.e18Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Without FA supplementation, and when PA levels at the INM are low (Figures 2E and 2F, control), the NLS-PA sensor exhibited a nucleoplasmic localization. Stearic acid (C18:0; 16 mM) produced a ∼4-fold increase of LD volume per cell compared with control cells without FA supplementation (Figure 2G). However, PA levels at the INM remained low (i.e., the NLS-PA sensor stayed mostly nucleoplasmic), and we did not detect nLDs. In contrast, oleic acid (C18:1; 16 mM) induced a ∼10-fold increase of LD volume per cell and generated both cLDs as well as nLDs (Figures 2E–2G). nLDs were present in ∼10% of cells under the conditions tested. Interestingly, linoleic acid, a UFA with two double bonds (C18:2; 16 mM), generated a similar amount of total LDs as oleic acid (C18:1), but essentially no nLDs, even though INM PA levels were increased. Linoleic acid failed to induce nLDs in the range of 3–16 mM, though the LD volume per cell was similar to that induced by oleic acid (Figures 2F and 2G). This result was unexpected because we assumed that the nLD/cLD ratio would remain constant irrespective of the FA class and that an increase of PA would be permissive for nLD formation. Thus, nLD synthesis does not scale with the total amount of LDs in cells but appears to be influenced by the type of FA that is supplied and additional factors besides a high INM PA content. Importantly, increasing the number of double bonds in FAs (i.e., linoleic acid) induces cLD biogenesis, yet, may suppress nLD synthesis. An overload of cells with dietary UFAs has a dual effect as it increases both the total FA content as well as PL unsaturation (Garbarino et al., 2009Garbarino J. Padamsee M. Wilcox L. Oelkers P.M. D'Ambrosio D. Ruggles K.V. Ramsey N. Jabado O. Turkish A. Sturley S.L. Sterol and diacylglycerol acyltransferase deficiency triggers fatty acid-mediated cell death.J. Biol. Chem. 2009; 284: 30994-31005Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Hence, it has remained unclear to what extent lipid unsaturation per se contributes to LD production. Ole1 overexpression provided us with the opportunity to selectively increase the intracellular UFA/SFA ratio. Strikingly, this led to a massive synthesis of LDs (Figure S2H) even though cells were not overfed with nutrient FAs. The amount of newly synthesized LDs correlated positively with Ole1 protein levels and mainly affected LD volume rather than LD number per cell (Figures S2I–S2K). Hence, introducing a single double bond at the C9 position is a potent driver of LD biogenesis. We then asked, in which region of the ER network these LDs are synthesized (i.e., INM versus ONM/peripheral ER). To do so, we determined whether LDs represent nLDs, cLDs, or both, by employing the NLS-PA sensor as an nLD marker together with BODIPY as a marker for all LDs in a cell. Notably, Ole1 overexpression increased the amount of cLDs but yielded essentially no nLDs (Figures 2H and 2I). We used ino4Δ cells as a benchmark for cells with a high nLD content, since blocking the Opi1-Ino2/4 transcriptional circuit is a strong stimulus for both cLD and nLD production (Romanauska and Köhler, 2018Romanauska A. Köhler A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets.Cell. 2018; 174: 700-715.e18Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). We detected nLDs as prominent BODIPY- and PA-positive nuclear structures in ∼32% of ino4Δ cells (Figures 2H and 2I). In contrast, Ole1 overexpression selectively increased cLD production. Hence, our data suggest that the conversion of endogenous SFAs into UFAs by Ole1 favors cLD over nLD synthesis similar to what we had observed when exogenously providing double-unsaturated FAs. To understand whether the balance of nLDs and cLDs is controlled by the Mga2 feedback circuit (Figure 1A), we tested whether Mga2 elicits the same phenotype as Ole1. We created an Mga2 version that lacks the ER-anchored TM domain (Mga2ΔTM) and is constitutively imported into the nucleus. Full-length Mga2, expressed from its endogenous promoter, localized to the entire ER network, and these cells contained few small LDs. In contrast, Mga2ΔTM was imported into the nucleus and induced LD production (Figure 3A; see Figure S2L for expression levels and S2M for LD quantification). This effect was also seen upon overexpression of the wild-type Mga2 from a stronger GPD promoter. Strikingly, the overexpression of Mga2ΔTM from the GPD promoter led to a massive accumulation of LDs, which turned yeast into adipocyte-like cells (Figure 3A). Using the LipSat sensors, we confirmed that lipids of the INM and ER/ONM were highly unsaturated under these conditions (Figures 3B–3E and S3A–S3D). Overexpression of other transcriptional targets of Mga2, such as the FA elongase ELO1, the LD-associated fatty acyl-CoA synthetase FAA4 (Kelley and Ideker, 2009Kelley R. Ideker T. Genome-wide fitness and expression profiling implicate Mga2 in adaptation to hydrogen peroxide.PLoS Genet. 2009; 5e1000488Crossref PubMed Scopus (45) Google Scholar), or the sterol-synthesis enzyme MVD1 (see transcriptome below), did not increase cellular LDs (Figure S2N). In contrast, overexpression of Spt23, the paralog of Mga2, did induce LDs (Figure S3E). Hence, Mga2/Spt23 overexpression resembles Ole1 overexpression. To quantitatively study the ultrastructure of LDs produced by the Mga2-Ole1 circuit, we employed transmission electron microscopy (TEM). As a control, we examined ino4Δ cells, which produce both nLDs and cLDs. With TEM we found nLDs in ∼23% of ino4Δ cells, but only in ∼2% of Mga2ΔTM-expressing cells (Figures 3G, 3H, and S3I). The TEM data are in agreement with live-cell imaging, in which we visualized the topological relationship of LDs with the NE by combining BODIPY staining with the INM marker Heh2-mCherry (Figures S3F and S3G). The ino4Δ cells contained nLDs in ∼18% of cells compared with only ∼5% of Mga2ΔTM-expressing cells. We previously described INM evaginations as a feature of nLD formation in ino4Δ cells (Romanauska and Köhler, 2018Romanauska A. Köhler A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets.Cell. 2018; 174: 700-715.e18Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) (Figure 3H). These INM evaginations were absent in Mga2ΔTM cells (Figures 3G and 3H). Importantly, the low number of nLDs in Mga2ΔTM cells is not caused by a reduced overall LD content because Mga2ΔTM cells contain a higher LD volume per cell than ino4Δ cells (Figure 3F). In sum, our data indicate that cells cope with lipid unsaturation by specifically producing cLDs rather than nLDs and that this bias is mediated by the Mga2/Ole1 pathway. To understand the mechanism by which cel