The Inner Nuclear Membrane Is a Metabolically Active Territory that Generates Nuclear Lipid Droplets.

The inner nuclear membrane (INM) encases the genome and is fused with the outer nuclear membrane (ONM) to form the nuclear envelope. The ONM is contiguous with the endoplasmic reticulum (ER), the main site of phospholipid synthesis. In contrast to the ER and ONM, evidence for a metabolic activity of the INM has been lacking. Here, we show that the INM is an adaptable membrane territory capable of lipid metabolism. S. cerevisiae cells target enzymes to the INM that can promote lipid storage. Lipid storage involves the synthesis of nuclear lipid droplets from the INM and is characterized by lipid exchange through Seipin-dependent membrane bridges. We identify the genetic circuit for nuclear lipid droplet synthesis and a role of these organelles in regulating this circuit by sequestration of a transcription factor. Our findings suggest a link between INM metabolism and genome regulation and have potential relevance for human lipodystrophy.

Fine-tuning phosphatidic acid production for optimal plant stress responses.

Phosphatidic acid (PA) is involved in biotic and abiotic stress responses in plants. Here, we summarize quantitative lipidomics and real-time imaging used in PA studies and highlight recent studies of diacylglycerol (DAG) kinase (DGK) 5, an enzyme involved in PA biosynthesis, facilitating fine-tuning PA production for optimal stress responses in plants.

Coupling lipid synthesis with nuclear envelope remodeling.

The nuclear envelope (NE) is a protective barrier to the genome, yet its membranes undergo highly dynamic remodeling processes that are necessary for cell growth and maintenance. While mechanisms by which proteins promote NE remodeling are emerging, the types of bilayer lipids and the lipid-protein interactions that define and sculpt nuclear membranes remain elusive. The NE is continuous with the endoplasmic reticulum (ER) and recent evidence suggests that lipids produced in the ER are harnessed to remodel nuclear membranes. In this review, we examine new roles for lipid species made proximally within the ER and locally at the NE to control NE dynamics. We further explore how the biosynthesis of lipids coordinates NE remodeling to ensure genome protection.

Endocannabinoid biosynthetic enzymes regulate pain response via LKB1-AMPK signaling.

Diacylglycerol lipase-beta (DAGLß) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLß ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLß in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLß blockade, thereby directly supporting DAGLß-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.

Diacylglycerol-dependent hexamers of the SNARE-assembling chaperone Munc13-1 cooperatively bind vesicles.

Munc13-1 is essential for vesicle docking and fusion at the active zone of synapses. Here, we report that Munc13-1 self-assembles into molecular clusters within diacylglycerol-rich microdomains present in phospholipid bilayers. Although the copy number of Munc13-1 molecules in these clusters has a broad distribution, a systematic Poisson analysis shows that this is most likely the result of two molecular species: monomers and mainly hexameric oligomers. Each oligomer is able to capture one vesicle independently. Hexamers have also been observed in crystals of Munc13-1 that form between opposed phospholipid bilayers [K. Grushin, R. V. Kalyana Sundaram, C. V. Sindelar, J. E. Rothman, Proc. Natl. Acad. Sci. U.S.A. 119, e2121259119 (2022)]. Mutations targeting the contacts stabilizing the crystallographic hexagons also disrupt the isolated hexamers, suggesting they are identical. Additionally, these mutations also convert vesicle binding from a cooperative to progressive mode. Our study provides an independent approach showing that Munc13-1 can form mainly hexamers on lipid bilayers each capable of vesicle capture.

Protein kinase C signaling "in" and "to" the nucleus: Master kinases in transcriptional regulation.

PKC is a multifunctional family of Ser-Thr kinases widely implicated in the regulation of fundamental cellular functions, including proliferation, polarity, motility, and differentiation. Notwithstanding their primary cytoplasmic localization and stringent activation by cell surface receptors, PKC isozymes impel prominent nuclear signaling ultimately impacting gene expression. While transcriptional regulation may be wielded by nuclear PKCs, it most often relies on cytoplasmic phosphorylation events that result in nuclear shuttling of PKC downstream effectors, including transcription factors. As expected from the unique coupling of PKC isozymes to signaling effector pathways, glaring disparities in gene activation/repression are observed upon targeting individual PKC family members. Notably, specific PKCs control the expression and activation of transcription factors implicated in cell cycle/mitogenesis, epithelial-to-mesenchymal transition and immune function. Additionally, PKCs isozymes tightly regulate transcription factors involved in stepwise differentiation of pluripotent stem cells toward specific epithelial, mesenchymal, and hematopoietic cell lineages. Aberrant PKC expression and/or activation in pathological conditions, such as in cancer, leads to profound alterations in gene expression, leading to an extensive rewiring of transcriptional networks associated with mitogenesis, invasiveness, stemness, and tumor microenvironment dysregulation. In this review, we outline the current understanding of PKC signaling "in" and "to" the nucleus, with significant focus on established paradigms of PKC-mediated transcriptional control. Dissecting these complexities would allow the identification of relevant molecular targets implicated in a wide spectrum of diseases.

Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis.

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

Catalytic core function of yeast Pah1 phosphatidate phosphatase reveals structural insight into its membrane localization and activity control.

The PAH1-encoded phosphatidate (PA) phosphatase is a major source of diacylglycerol for the production of the storage lipid triacylglycerol and a key regulator for the de novo phospholipid synthesis in Saccharomyces cerevisiae. The catalytic function of Pah1 depends on its membrane localization which is mediated through its phosphorylation by multiple protein kinases and dephosphorylation by the Nem1-Spo7 protein phosphatase complex. The full-length Pah1 is composed of a catalytic core (N-LIP and HAD-like domains, amphipathic helix, and the WRDPLVDID domain) and non-catalytic regulatory sequences (intrinsically disordered regions, RP domain, and acidic tail) for phosphorylation and interaction with Nem1-Spo7. How the catalytic core regulates Pah1 localization and cellular function is not clear. In this work, we analyzed a variant of Pah1 (i.e., Pah1-CC (catalytic core)) that is composed only of the catalytic core. Pah1-CC expressed on a low-copy plasmid complemented the pah1Δ mutant phenotypes (e.g., nuclear/ER membrane expansion, reduced levels of triacylglycerol, and lipid droplet formation) without requiring Nem1-Spo7. The cellular function of Pah1-CC was supported by its PA phosphatase activity mostly associated with the membrane fraction. Although functional, Pah1-CC was distinct from Pah1 in the protein and enzymological properties, which include overexpression toxicity, association with heat shock proteins, and significant reduction of the Vmax value. These findings on the Pah1 catalytic core enhance the understanding of its structural requirements for membrane localization and activity control.

The Saccharomyces cerevisiae Spo7 basic tail is required for Nem1-Spo7/Pah1 phosphatase cascade function in lipid synthesis.

The Saccharomyces cerevisiae Nem1-Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1-Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1-Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1-3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1-Spo7 complex-mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

Protein kinase Hsl1 phosphorylates Pah1 to inhibit phosphatidate phosphatase activity and regulate lipid synthesis in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids. The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues. Here, we established Pah1 as a bona fide substrate of septin-associated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances the understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy.

Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial-associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+ ) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta-b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2-mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA-mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.

Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis.

Nuclear envelope (NE) expansion must be controlled to maintain nuclear shape and function. The nuclear membrane expands massively during closed mitosis, enabling chromosome segregation within an intact NE. Phosphatidic acid (PA) and diacylglycerol (DG) can both serve as biosynthetic precursors for membrane lipid synthesis. How they are regulated in time and space and what the implications are of changes in their flux for mitotic fidelity are largely unknown. Using genetically encoded PA and DG probes, we show that DG is depleted from the inner nuclear membrane during mitosis in the fission yeast Schizosaccharomyces pombe, but PA does not accumulate, indicating that it is rerouted to membrane synthesis. We demonstrate that DG-to-PA conversion catalyzed by the diacylglycerol kinase Dgk1 (also known as Ptp4) and direct glycerophospholipid synthesis from DG by diacylglycerol cholinephosphotransferase/ethanolaminephosphotransferase Ept1 reinforce NE expansion. We conclude that DG consumption through both the de novo pathway and the Kennedy pathway fuels a spike in glycerophospholipid biosynthesis, controlling NE expansion and, ultimately, mitotic fidelity.

CdsA, a CDP-diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons.

CdsA is a CDP-diacylglycerol synthase essential for phospholipid and glycolipid MPIase biosynthesis, and therefore for growth. The initiation codon of CdsA has been assigned as "TTG," while methionine at the 37th codon was reported to be an initiation codon in the original report. Since a vector containing the open reading frame starting with "TTG" under a controllable promoter complemented the cdsA knockout, "TTG" could function as an initiation codon. However, no evidence supporting that this "TTG" is the sole initiation codon has been reported. We determined the initiation codon by examining the ability of mutants around the N-terminal region to complement cdsA mutants. Even if the "TTG" was substituted with a stop codon, the clear complementation was observed. Moreover, the clones with multiple mutations of stop codons complemented the cdsA mutant up to the 37th codon, indicating that cdsA possesses multiple codons that can function as initiation codons. We constructed an experimental system in which the chromosomal expression of cdsA can be analyzed. By means of this system, we found that the cdsA mutant with substitution of "TTG" with a stop codon is fully functional. Thus, we concluded that CdsA contains multiple initiation codons.

Glycoursodeoxycholic Acid Alleviates Arterial Thrombosis via Suppressing Diacylglycerol Kinases Activity in Platelet.

BACKGROUND: Glycoursodeoxycholic acid (GUDCA) has been acknowledged for its ability to regulate lipid homeostasis and provide benefits for various metabolic disorders. However, the impact of GUDCA on arterial thrombotic events remains unexplored. The objective of this study is to examine the effects of GUDCA on thrombogenesis and elucidate its underlying mechanisms. METHODS: Plasma samples from patients with arterial thrombotic events and diet-induced obese mice were collected to determine the GUDCA concentrations using mass spectrometry. Multiple in vivo murine thrombosis models and in vitro platelet functional assays were conducted to comprehensively evaluate the antithrombotic effects of GUDCA. Moreover, lipidomic analysis was performed to identify the alterations of intraplatelet lipid components following GUDCA treatment. RESULTS: Plasma GUDCA level was significantly decreased in patients with arterial thrombotic events and negatively correlated with thrombotic propensity in diet-induced obese mice. GUDCA exhibited prominent suppressing effects on platelet reactivity as evidenced by the attenuation of platelet activation, secretion, aggregation, spreading, and retraction (P<0.05). In vivo, GUDCA administration robustly alleviated thrombogenesis (P<0.05) without affecting hemostasis. Mechanistically, GUDCA inhibited DGK (diacylglycerol kinase) activity, leading to the downregulation of the phosphatidic acid-mediated signaling pathway. Conversely, phosphatidic acid supplementation was sufficient to abolish the antithrombotic effects of GUDCA. More importantly, long-term oral administration of GUDCA normalized the enhanced DGK activity, thereby remarkably alleviating the platelet hyperreactivity as well as the heightened thrombotic tendency in diet-induced obese mice (P<0.05). CONCLUSIONS: Our study implicated that GUDCA reduces platelet hyperreactivity and improves thrombotic propensity by inhibiting DGKs activity, which is a potentially effective prophylactic approach and promising therapeutic agent for arterial thrombotic events.

Tumor-associated macrophage (TAM)-secreted CCL22 confers cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells via regulating the activity of diacylglycerol kinase α (DGKα)/NOX4 axis.

Tumor-associated macrophages (TAMs) are often associated with chemoresistance and resultant poor clinical outcome in solid tumors. Here, we demonstrated that TAMs-released chemokine-C-C motif chemokine 22 (CCL22) in esophageal squamous cell carcinoma (ESCC) stroma was tightly correlated with the chemoresistance of ESCC patients. TAMs-secreted CCL22 was able to block the growth inhibitory and apoptosis-promoting effects of cisplatin on ESCC cells. Mechanistically, CCL22 stimulated intratumoral diacylglycerol kinase α (DGKα) to produce phosphatidic acid (PA), which suppressed the activity of NADPH oxidase 4 (NOX4) and then blocked the overproduction of intratumoral reactive species oxygen (ROS) induced by cisplatin. CCL22 activated DGKα/nuclear factor-κB (NF-κB) axis to upregulate the level of several members of ATP binding cassette (ABC) transporter superfamily, including ABC sub-family G member 4 (ABCG4), ABC sub-family A member 3 (ABCA3), and ABC sub-family A member 5 (ABCA5), to lower the intratumoral concentration of cisplatin. Consequently, these processes induced the cisplatin resistance in ESCC cells. In xenografted models, targeting DGKα with 5'-cholesterol-conjugated small-interfering (si) RNA enhanced the chemosensitivity of cisplatin in ESCC treatment, especially in the context of TAMs. Our data establish the correlation between the TAMs-induced intratumoral metabolic product/ROS axis and chemotherapy efficacy in ESCC treatment and reveal relevant molecular mechanisms.

Diacylglycerol kinase-ε is S-palmitoylated on cysteine in the cytoplasmic end of its N-terminal transmembrane fragment.

Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

Saturated fatty acid- and/or monounsaturated fatty acid-containing phosphatidic acids selectively interact with heat shock protein 27.

Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T-cell activity attenuation, preferentially produces saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs), such as 16:0/16:0-, 16:0/18:0-, and 16:1/16:1-PA, in melanoma cells. In the present study, we searched for the target proteins of 16:0/16:0-PA in melanoma cells and identified heat shock protein (HSP) 27, which acts as a molecular chaperone and contributes to cancer progression. HSP27 more strongly interacted with PA than other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. Moreover, HSP27 is more preferentially bound to SFA- and/or MUFA-containing PAs, including 16:0/16:0- and 16:0/18:1-PAs, than PUFA-containing PAs, including 18:0/20:4- and 18:0/22:6-PA. Furthermore, HSP27 and constitutively active DGKα expressed in COS-7 cells colocalized in a DGK activity-dependent manner. Notably, 16:0/16:0-PA, but not phosphatidylcholine or 16:0/16:0-phosphatidylserine, induced oligomer dissociation of HSP27, which enhances its chaperone activity. Intriguingly, HSP27 protein was barely detectable in Jurkat T cells, while the protein band was intensely detected in AKI melanoma cells. Taken together, these results strongly suggest that SFA- and/or MUFA-containing PAs produced by DGKα selectively target HSP27 and regulate its cancer-progressive function in melanoma cells but not in T cells.

Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway.

Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.

Phosphatidate phosphatase Pah1 contains a novel RP domain that regulates its phosphorylation and function in yeast lipid synthesis.

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The enzyme controls whether cells utilize PA to produce membrane phospholipids or the major storage lipid triacylglycerol. PA levels, which are regulated by the enzyme reaction, also control the expression of UASINO-containing phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit. Pah1 function is largely controlled by its cellular location, which is mediated by phosphorylation and dephosphorylation. Multiple phosphorylations sequester Pah1 in the cytosol and protect it from 20S proteasome-mediated degradation. The endoplasmic reticulum-associated Nem1-Spo7 phosphatase complex recruits and dephosphorylates Pah1 allowing the enzyme to associate with and dephosphorylate its membrane-bound substrate PA. Pah1 contains domains/regions that include the N-LIP and haloacid dehalogenase-like catalytic domains, N-terminal amphipathic helix for membrane binding, C-terminal acidic tail for Nem1-Spo7 interaction, and a conserved tryptophan within the WRDPLVDID domain required for enzyme function. Through bioinformatics, molecular genetics, and biochemical approaches, we identified a novel RP (regulation of phosphorylation) domain that regulates the phosphorylation state of Pah1. We showed that the ΔRP mutation results in a 57% reduction in the endogenous phosphorylation of the enzyme (primarily at Ser-511, Ser-602, and Ser-773/Ser-774), an increase in membrane association and PA phosphatase activity, but reduced cellular abundance. This work not only identifies a novel regulatory domain within Pah1 but emphasizes the importance of the phosphorylation-based regulation of Pah1 abundance, location, and function in yeast lipid synthesis.

Conserved regions of the regulatory subunit Spo7 are required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid synthesis.

In the yeast Saccharomyces cerevisiae, the Nem1-Spo7 complex is a protein phosphatase that activates Pah1 phosphatidate phosphatase at the nuclear-endoplasmic reticulum membrane for the synthesis of triacylglycerol. The Nem1-Spo7/Pah1 phosphatase cascade largely controls whether phosphatidate is partitioned into the storage lipid triacylglycerol or into membrane phospholipids. The regulated synthesis of the lipids is crucial for diverse physiological processes during cell growth. Spo7 in the protein phosphatase complex is required as a regulatory subunit for the Nem1 catalytic subunit to dephosphorylate Pah1. The regulatory subunit contains three conserved homology regions (CR1, CR2, and CR3). Previous work showed that the hydrophobicity of LLI (residues 54-56) within CR1 is important for Spo7 function in the Nem1-Spo7/Pah1 phosphatase cascade. In this work, by deletion and site-specific mutational analyses, we revealed that CR2 and CR3 are also required for Spo7 function. Mutations in any one of the conserved regions were sufficient to disrupt the function of the Nem1-Spo7 complex. We determined that the uncharged hydrophilicity of STN (residues 141-143) within CR2 was required for Nem1-Spo7 complex formation. In addition, the hydrophobicity of LL (residues 217 and 219) within CR3 was important for Spo7 stability, which indirectly affected complex formation. Finally, we showed the loss of Spo7 CR2 or CR3 function by the phenotypes (e.g., reduced amounts of triacylglycerol and lipid droplets, temperature sensitivity) that are attributed to defects in membrane translocation and dephosphorylation of Pah1 by the Nem1-Spo7 complex. These findings advance knowledge of the Nem1-Spo7 complex and its role in lipid synthesis regulation.