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Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate the uptake and synthesis of cholesterol and fatty acids in mammalian cells. SREBP cleavage-activating protein (SCAP) is an endoplasmic reticulum (ER) protein that binds newly synthesized SREBP, retaining it in the ER where SREBP is inactive. SCAP binds cholesterol and functions as the cholesterol sensor in this regulatory system. Under low cholesterol conditions, SCAP escorts SREBP from the ER to the Golgi apparatus where two proteases sequentially cleave and activate SREBP. Given their central importance in maintaining cellular lipid homeostasis, other mechanisms exist to regulate SREBP activity, such as control of protein synthesis and degradation. Here, we describe methods to assay ER-to-Golgi transport of SCAP in vitro using immunofluorescence microscopy and two different cell systems, Chinese hamster ovary (CHO) cells stably expressing hamster GFP-SCAP and human HeLa cells transiently expressing human GFP-SCAP. These methods will permit investigators to determine if cellular perturbations act by affecting the ER-to-Golgi transport of SCAP.

Progesterone Receptor Membrane Component 1 (PGRMC1) is a heme-binding protein that has been implicated in a wide range of cell and tissue functions, including cytochromes P450 activity, heme homeostasis, cancer, female reproduction, and protein quality control. Despite an extensive body of literature, a relative lack of mechanistic insight means that how PGRMC1 functions in these different aspects of biology is largely unknown. This review provides an overview of the PGRMC1 literature, highlighting what information is rigorously supported by experimental evidence and where additional investigation is warranted. The central role of PGRMC1 in supporting cytochrome P450 activity is discussed at length. Building on existing models of PGRMC1 function, a speculative model is proposed using the reviewed literature in which PGRMC1 functions as a heme chaperone to shuttle heme from its site of synthesis in the mitochondrion to other subcellular compartments. By spotlighting knowledge gaps, this review will motivate investigators to better understand this enigmatic protein.

Fission yeast Dap1 is a membrane-bound, heme binding protein that binds directly to both fission yeast cytochromes P450, Erg5 and Erg11, and is required for Erg5 and Erg11 function in ergosterol synthesis (Hughes et al., 2007). Progesterone receptor membrane component 1 (PGRMC1) is a mammalian homolog of Dap1 that is also required for cytochromes P450 activity (Hughes et al., 2007; Mallory et al., 2005; McGuire et al., 2021). In mouse liver, PGRMC1 binds more than 13 cytochromes P450 and supports their stability and consequently P450 activity (McGuire et al., 2021). Interestingly, PGRMC2, a paralog of PGRMC1, also binds heme and functions as a heme chaperone required to deliver heme from the mitochondrion to the nucleus in mice (Galmozzi et al., 2019). Whether PGRMC1 also functions as a heme chaperone and what specific role PGRMC1 heme binding serves are unknown.

Lipid metabolic reprogramming is a hallmark of cancer that contributes to tumorigenesis by supporting energy metabolism and the synthesis of signaling molecules, cellular membranes, and protein modifications. Cells modulate lipid metabolic genes through the regulation of sterol regulatory element-binding proteins (SREBPs), which serve as transcriptional activators of genes responsible for fatty acid and cholesterol synthesis and uptake. Despite efforts demonstrating that the SREBP pathway is an attractive chemotherapeutic target, a potent bioavailable inhibitor of the pathway has not yet been discovered. Here, we identify druggable SREBP target genes as potential targets in pancreatic ductal adenocarcinoma (PDAC). Results: To discover candidate genes, we constructed a CRISPR knockout library containing 148 known SREBP target genes. We used a patient-derived PDAC cell line to conduct the knockout screens in vivo (n = 7 tumors) by injecting library-infected cells into the pancreas of nude mice. After 4 weeks of growth, mouse PDAC tumors showed a statistically significant depletion of guide RNAs targeting 8 genes within the mevalonate pathway, which is responsible for synthesizing cholesterol and isoprenoids such as farnesyl-diphosphate and geranyl-diphosphate. One of the most significant hits was HMG-CoA reductase (HMGCR), which catalyzes the formation of mevalonate. We chose to investigate statins, which are a well-characterized class of FDA-approved drugs that inhibit HMGCR. Using flow cytometry, we stained for annexin-V as a marker for apoptosis and found minimal cell death induced by Fluvastatin alone. Given that inhibiting HMGCR would disrupt lipid homeostasis and in turn activate the SREBP pathway, we hypothesized that a combination therapy with an SREBP inhibitor, TMDP, would potentiate statin-induced cell death. We observed minimal cell death with TMDP alone, but striking synergy between Fluvastatin and TMDP, showing a five-fold increase in annexin-V staining after 48 hours. Mevalonate and geranylgeranyl-diphosphate both fully rescued this cell death to basal levels. Conclusions: In vivo CRISPR screening is a powerful tool for the identification of novel therapeutic targets for cancer. We found that the mevalonate pathway plays a significant role in pancreatic tumor growth in vivo and that existing FDA-approved drugs can be used in combination with an SREBP inhibitor to dramatically enhance cell killing in vitro. Citation Format: Casie S. Kubota, Chiaki T. Ishida, Stephanie Myers, Peter J. Espenshade. Identifying druggable lipid synthesis targets as novel therapeutics for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B011.

Sterol Regulatory Element-Binding Protein-1c (SREBP-1c) is translated as an inactive precursor-protein that is proteolytically activated to promote fatty-acid (FA) biosynthesis, when unsaturated (u)FAs are scarce. During fasting, however, lipogenesis is low, and adipose-tissue lipolysis supplies the organism with FAs. Adipose TriGlyceride Lipase (ATGL) is the rate-limiting enzyme for lipolysis, and it preferentially hydrolyzes uFAs. Therefore, we hypothesized that ATGL-derived FAs may suppress the proteolytic activation of SREBP-1c in the liver. Here we show that (i) SREBP-1c is inactive during fasting but active after refeeding, (ii) uFA species liberated by ATGL suppress SREBP-1c activation in vitro, (iii) SREBP-1c is hyper-activated in livers of mice lacking ATGL, and (iv) pharmacological inhibition of ATGL selectively activates SREBP-1c in hepatocytes. Our findings highlight an ATGL/SREBP-1c axis, instrumental to coordinate lipogenesis and lipolysis, whose homeostatic regulation is crucial to avoid severe diseases including diabetes, cardiomyopathy, and even cancer.

Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. Whereas much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is un-known. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an independent regulator of HIF. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of the HIFα subunit of these heterodimeric transcription factors comparable with hypoxia with activation of downstream target genes. The addition of fatty acids to culture medium suppressed this signal, which required an intact mitochondrial respiratory chain. Mechanistically, fatty acids and oxygen are distinct signals integrated to control HIF activity. Finally, we observed lipid signaling to HIF and changes in target gene expression in developing zebrafish and adult mice, and this pathway operates in cancer cells from a range of tissues. This study identifies fatty acids as a physiological modulator of HIF, defining a mechanism for lipoprotein regulation that functions in parallel to oxygen.

Low oxygen adaptation is essential for aerobic fungi that must survive in varied oxygen environments. Pathogenic fungi in particular must adapt to the low oxygen host tissue environment in order to cause infection. Maintenance of lipid homeostasis is especially important for cell growth and proliferation, and is a highly oxygen-dependent process. In this review, we focus on recent advances in our understanding of the transcriptional regulation and coordination of the low oxygen response across fungal species, paying particular attention to pathogenic fungi. Comparison of lipid homeostasis pathways in these organisms suggests common mechanisms of transcriptional regulation and points toward untapped potential to target low oxygen adaptation in antifungal development.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with limited diagnostic and therapeutic options. Due to its proliferative nature and thick, desmoplastic stroma, PDAC tumor cells are challenged with meeting a high demand for lipids in a hypoxic, lipid-poor environment. Cancer cells respond to lipid conditions through sterol regulatory element-binding proteins (SREBPs). SREBPs are master transcriptional regulators of lipid homeostasis and require SREBP cleavage activating protein (SCAP) during signaling. While the SREBP pathway has been implicated in several cancers, its role in PDAC has not been examined. In this study, we assess the requirement of this pathway using both in vitro and in vivo model systems. Furthermore, we target the SREBP pathway using a novel combination therapy of two FDA-approved compounds: dipyridamole and statins. Methods: We acquired four patient-derived pancreatic adenocarcinoma cell lines containing mutations in both Kras and p53 and knocked out SCAP in each cell line followed by gene-based rescue. Functional growth assays were performed in both lipid-poor and lipid-rich media conditions. Subcutaneous and pancreatic orthotopic xenografts were performed in nude mice using these same cell lines. A previously established mouse line, LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1 Cre (KPC) on a C57Bl/6 background, was utilized as a PDAC model. KPC mice lacking Scap in one or both alleles (Sfl/+ or Sfl/fl) were generated. Cohort survival and histologic analysis were performed for all mice, and results plotted on a Kaplan-Meier survival curve. In each patient cell line, the following drugs were applied individually and in combination: Dipyridamole (DP), tetramethyl-DP (TMDP), Fluvastatin, and Simvastatin. Cell viability post-treatment was assessed, and synergy calculated for each combination using SynergyFinder. Results: In lipid-poor conditions, SCAP knockout cells showed significantly reduced growth when compared to wild type or SCAP rescued cells. In tumor xenograft models, SCAP knockout cells exhibited reduced tumor growth and tumor volume when compared to wild-type cells. In the genetically engineered mouse models, KPC mice exhibited a median survival time of 289 days, while KPCSfl/+ mice show a significantly increased median survival time of 425 days. Additionally, KPCSfl/fl mice did not develop invasive PDAC. Cells treated individually with DP, TMDP and both statins exhibit lipid-dependent growth defects in all cell lines tested. In combination, DP with either statin as well as TMDP with either statin demonstrate synergy in lipid-poor conditions. Conclusions: Our results demonstrate that loss of SCAP in PDAC tumor cells alters the growth capability both in vitro and using mouse xenograft models. Additionally, heterozygous loss of Scap in the KPC mouse model significantly increased survival. Finally, dipyridamole works in synergy with statins to alter growth of PDAC tumor cells. These findings suggest that targeting the SREBP pathway has significant therapeutic potential in PDAC. Citation Format: Stephanie Myers, Meredith McGuire, Wei Shao, Chine Liu, Theodore Ewachiw, Zeshaan Rasheed, William Matsui, Toni Sepalla, Richard Burkhart, Peter Espenshade. Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-009.

Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally. In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes.

Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA+) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe, generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe.

The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2. Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis.

Data on the perceptions of scientists suggest a moderate public distrust of scientist's motivations. Bettridge et al. suggest scientist's reluctance to engage the public on controversial ethical issues may be a contributing factor. The authors propose a Scientist's Oath to send a clear message to the public about our ideals.

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipid homeostasis and activate expression of genes required for fatty acid, triglyceride, and cholesterol synthesis and uptake. SREBP cleavage activating protein (SCAP) plays an essential role in SREBP activation by mediating endoplasmic reticulum (ER)-to-Golgi transport of SREBP. In the Golgi, membrane-bound SREBPs are cleaved sequentially by the site-1 and site-2 proteases. Recent studies have shown a requirement for the SREBP pathway in the development of fatty liver disease and tumor growth, making SCAP a target for drug development. Fatostatin is a chemical inhibitor of the SREBP pathway that directly binds SCAP and blocks its ER-to-Golgi transport. In this study, we determined that fatostatin blocks ER exit of SCAP and showed that inhibition is independent of insulin-induced gene proteins, which function to retain the SCAP-SREBP complex in the ER. Fatostatin potently inhibited cell growth, but unexpectedly exogenous lipids failed to rescue proliferation of fatostatin-treated cells. Furthermore, fatostatin inhibited growth of cells lacking SCAP. Using a vesicular stomatitis virus glycoprotein (VSVG) trafficking assay, we demonstrated that fatostatin delays ER-to-Golgi transport of VSVG. In summary, fatostatin inhibited SREBP activation, but fatostatin additionally inhibited cell proliferation through both lipid-independent and SCAP-independent mechanisms, possibly by general inhibition of ER-to-Golgi transport.

Prolyl hydroxylation functions in diverse cellular pathways, such as collagen biogenesis, oxygen sensing, and translation termination. Prolyl hydroxylation is catalyzed by 2-oxoglutarate (2-OG) oxygenases. The fission yeast 2-OG oxygenase Ofd1 dihydroxylates the 40S ribosomal protein Rps23 and regulates the hypoxic response by controlling activity and stability of the sterol regulatory element-binding protein Sre1. Multiple substrates have been found for 2-OG oxygenases, yet the only known substrate of Ofd1 and its homologs is Rps23. Here, we report the first fission yeast prolyl hydroxylome and demonstrate that hydroxylation is more prevalent than previously known. Using quantitative mass spectrometry, we identify Rpb10, a shared subunit in RNA polymerase I, II, and III, as a novel Ofd1 substrate. In addition, we discovered six Ofd1 binding partners and 16 additional Ofd1 candidate substrates. Although Ofd1 promotes Sre1 degradation, proteomic analysis revealed that Ofd1 does not broadly regulate protein degradation. Instead, the effect of Ofd1 on the proteome is through negative regulation of Sre1N. Finally, we show that the interaction between Ofd1 and the Sre1 bHLH region is conserved across Sre1 homologs suggesting that Ofd1-dependent regulation of SREBPs may be conserved in other fungi. Collectively, these studies provide a new dataset of post-translational modifications and expand the biological functions for a conserved prolyl hydroxylase.

Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1–Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking. Background: Proteolytic activation of fungal SREBP requires the five-subunit Golgi Dsc E3 ligase. Results: Dsc1 is an active E3 ligase; loss of Dsc E3 ligase activity leads to ER localization of the Dsc complex. Conclusion: ER exit of the Dsc E3 ligase requires E3 ligase activity. Significance: This is the first example of enzyme activity-dependent protein sorting in the secretory pathway.

The sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) are central players in the SREBP pathway, which control the cellular lipid homeostasis. SCAP binds to SREBP through their carboxyl (C) domains and escorts SREBP from the endoplasmic reticulum to the Golgi upon sterol depletion. A conserved pathway, with the homologues of SREBP and SCAP being Sre1 and Scp1, was identified in fission yeast Schizosaccharomyces pombe. Here we report the in vitro reconstitution of the complex between the C domains of Sre1 and Scp1 as well as the crystal structure of the WD40 domain of Scp1 at 2.1 A resolution. The structure reveals an eight-bladed β-propeller that exhibits several distinctive features from a canonical WD40 repeat domain. Structural and biochemical characterization led to the identification of two Scp1 elements that are involved in Sre1 recognition, an Arg/Lys-enriched surface patch on the top face of the WD40 propeller and a 30-residue C-terminal tail. The structural and biochemical findings were corroborated by in vivo examinations. These studies serve as a framework for the mechanistic understanding and further functional characterization of the SREBP and SCAP proteins in fission yeast and higher organisms.

The prolyl-3,4-dihydroxylase Ofd1 and nuclear import adaptor Nro1 regulate the hypoxic response in fission yeast by controlling activity of the sterol regulatory element-binding protein transcription factor Sre1. Here, we identify an extra-ribosomal function for uS12/Rps23 central to this regulatory system. Nro1 binds Rps23, and Ofd1 dihydroxylates Rps23 P62 in complex with Nro1. Concurrently, Nro1 imports Rps23 into the nucleus for assembly into 40S ribosomes. Low oxygen inhibits Ofd1 hydroxylase activity and stabilizes the Ofd1-Rps23-Nro1 complex, thereby sequestering Ofd1 from binding Sre1, which is then free to activate hypoxic gene expression. In vitro studies demonstrate that Ofd1 directly binds Rps23, Nro1, and Sre1 through a consensus binding sequence. Interestingly, Rps23 expression modulates Sre1 activity by changing the Rps23 substrate pool available to Ofd1. To date, oxygen is the only known signal to Sre1, but additional nutrient signals may tune the hypoxic response through control of unassembled Rps23 or Ofd1 activity., eLife digest Animals, plants, and fungi need oxygen to release energy within their cells and for other chemical reactions. Enzymes that use oxygen typically become less active when less oxygen is available, and this makes them well suited to help cells sense oxygen. These enzymes include oxygenases, some of which modify proteins by adding oxygen to specific sites in a reaction called hydroxylation. Oxygenases control how mammals adapt to low levels of oxygen – a condition referred to as hypoxia. These enzymes achieve this by hydroxylating a protein – specifically a transcription factor – that turns on genes for survival in low oxygen. Cells quickly destroy the hydroxylated transcription factor but when oxygen is limiting, it remains unmodified. This means that, rather than being destroyed, the transcription factor binds DNA, and activates genes that keep the cells alive and growing in low oxygen. In fission yeast, an oxygenase called Ofd1 controls the activity of a transcription factor called Sre1. Yeast requires Sre1 to grow when oxygen is limiting. Exactly how Ofd1 regulates Sre1 is unknown, but the mechanism is different from that in mammals because regulation of gene expression does not need Sre1 to be hydroxylated. Now, Clasen et al. report that Ofd1 actually hydroxylates another protein called Rps23. This protein is one of about 80 that form the cell’s protein-building machinery, the ribosome. It turns out that, before Rps23 becomes part of the ribosome, it binds Ofd1 in a complex with other proteins. The multi-protein complex then acts to hydroxylate and transport Rps23 into the nucleus, where ribosomes are built and where the cell stores its DNA. When little oxygen is around, Ofd1 cannot hydroxylate Rps23. This stops the complex from falling apart and traps Ofd1 away from the transcription factor Sre1. When not bound by Ofd1, Sre1 is free to turn on genes that allow growth at low levels of oxygen. Finally, Clasen et al. show that more unassembled Rps23 means less Ofd1 is available to inhibit Sre1, which controls the yeast cell’s response to hypoxia. Humans have proteins similar to Ofd1 and Rps23. As such, this pathway for sensing oxygen in yeast may occur in humans too. Further work is now needed to explore if other enzymes that hydroxylate ribosomal proteins work in a similar way.

Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires endoplasmic reticulum (ER)-resident, multi-subunit E3 ubiquitin ligases that function in ER-associated degradation. Using fission yeast, our lab identified the Golgi Dsc E3 ligase as required for proteolytic activation of fungal sterol regulatory element-binding protein transcription factors. The Dsc E3 ligase contains five integral membrane subunits and structurally resembles ER-associated degradation E3 ligases. Saccharomyces cerevisiae codes for homologs of Dsc E3 ligase subunits, including the Dsc1 E3 ligase homolog Tul1 that functions in Golgi protein quality control. Interestingly, S. cerevisiae lacks sterol regulatory element-binding protein homologs, indicating that novel Tul1 E3 ligase substrates exist. Here, we show that the S. cerevisiae Tul1 E3 ligase consists of Tul1, Dsc2, Dsc3, and Ubx3 and define Tul1 complex architecture. Tul1 E3 ligase function required each subunit as judged by vacuolar sorting of the artificial substrate Pep12D. Genetic studies demonstrated that Tul1 E3 ligase was required in cells lacking the multivesicular body pathway and under conditions of ubiquitin depletion. To identify candidate substrates, we performed quantitative diGly proteomics using stable isotope labeling by amino acids in cell culture to survey ubiquitylation in wild-type and tul1Δ cells. We identified 3116 non-redundant ubiquitylation sites, including 10 sites in candidate substrates. Quantitative proteomics found 4.5% of quantified proteins (53/1172) to be differentially expressed in tul1Δ cells. Correcting the diGly dataset for these differences increased the number of Tul1-dependent ubiquitylation sites. Together, our data demonstrate that the Tul1 E3 ligase functions in protein homeostasis under non-stress conditions and support a role in protein quality control. This quantitative diGly proteomics methodology will serve as a robust platform for screening for stress conditions that require Tul1 E3 ligase activity.

Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipogenesis. Release of membrane-bound SREBP requires SREBP cleavage-activating protein (SCAP) to escort SREBP from the endoplasmic reticulum (ER) to the Golgi for cleavage by site-1 and site-2 proteases. SCAP then recycles to the ER for additional rounds of SREBP binding and transport. Mechanisms regulating ER-to-Golgi transport of SCAP-SREBP are understood in molecular detail, but little is known about SCAP recycling. Here, we have demonstrated that SCAP Golgi-to-ER transport requires cleavage of SREBP at site-1. Reductions in SREBP cleavage lead to SCAP degradation in lysosomes, providing additional negative feedback control to the SREBP pathway. Current models suggest that SREBP plays a passive role prior to cleavage. However, we show that SREBP actively prevents premature recycling of SCAP-SREBP until initiation of SREBP cleavage. SREBP regulates SCAP in human cells and yeast, indicating that this is an ancient regulatory mechanism.

Sterol regulatory element-binding proteins (SREBPs) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain, thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2 and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis. Background: Yeast sterol regulatory element-binding proteins (SREBPs) are proteolytically activated by a mechanism distinct from that of mammals. Results: Site-directed mutagenesis studies define structural requirements for yeast SREBP cleavage. Conclusion: Yeast SREBP cleavage requires a novel conserved glycine-leucine motif. Significance: Understanding SREBP structural requirements aids mechanistic dissection of SREBP cleavage essential for fungal pathogenesis.

The engineered ascorbate peroxidase (APEX2) has been effectively employed in mammalian cells to identify protein–protein interactions. APEX2 fused to a protein of interest covalently tags nearby proteins with biotin-phenol (BP) when H2O2 is added to the cell culture medium. Subsequent affinity purification of biotinylated proteins allows for identification by MS. BP labelling occurs in 1 min, providing temporal control of labelling. The APEX2 tool enables proteomic mapping of subcellular compartments as well as identification of dynamic protein complexes, and has emerged as a new methodology for proteomic analysis. Despite these advantages, a related APEX2 approach has not been developed for yeast. Here we report methods to enable APEX2-mediated biotin labelling in yeast. Our work demonstrated that high osmolarity and disruption of cell wall integrity permits live-cell biotin labelling in Schizosaccharomyces pombe and Saccharomyces cerevisiae respectively. Under these conditions, APEX2 permitted targeted and proximity-dependent labelling of proteins. The methods described herein set the stage for large-scale proteomic studies in yeast. With modifications, the method is also expected to be effective in other organisms with cell walls, such as bacteria and plants.

Eukaryotic lipid synthesis is oxygen-dependent with cholesterol synthesis requiring 11 oxygen molecules and fatty acid desaturation requiring 1 oxygen molecule per double bond. Accordingly, organisms evaluate oxygen availability to control lipid homeostasis. The sterol regulatory element-binding protein (SREBP) transcription factors regulate lipid homeostasis. In mammals, SREBP-2 controls cholesterol biosynthesis, whereas SREBP-1 controls triacylglycerol and glycerophospholipid biosynthesis. In the fission yeast Schizosaccharomyces pombe, the SREBP-2 homolog Sre1 regulates sterol homeostasis in response to changing sterol and oxygen levels. However, notably missing is an SREBP-1 analog that regulates triacylglycerol and glycerophospholipid homeostasis in response to low oxygen. Consistent with this, studies have shown that the Sre1 transcription factor regulates only a fraction of all genes up-regulated under low oxygen. To identify new regulators of low oxygen adaptation, we screened the S. pombe nonessential haploid deletion collection and identified 27 gene deletions sensitive to both low oxygen and cobalt chloride, a hypoxia mimetic. One of these genes, mga2, is a putative transcriptional activator. In the absence of mga2, fission yeast exhibited growth defects under both normoxia and low oxygen conditions. Mga2 transcriptional targets were enriched for lipid metabolism genes, and mga2Δ cells showed disrupted triacylglycerol and glycerophospholipid homeostasis, most notably with an increase in fatty acid saturation. Indeed, addition of exogenous oleic acid to mga2Δ cells rescued the observed growth defects. Together, these results establish Mga2 as a transcriptional regulator of triacylglycerol and glycerophospholipid homeostasis in S. pombe, analogous to mammalian SREBP-1.

Sterol regulatory element-binding protein (SREBP) transcription factors regulate cellular lipogenesis and lipid homeostasis. Recent studies reveal expanding roles for SREBPs with the description of new regulatory mechanisms, the identification of unexpected transcriptional targets, and the discovery of functions for SREBPs in type II diabetes, cancer, immunity, neuroprotection, and autophagy.

The prolyl hydroxylase Ofd1 enables yeast cells to adapt to hypoxia by regulating the DNA binding and degradation of the hypoxic transcription factor Sre1N. These two regulatory functions are inseparable in vivo. A mathematical model of the Ofd1 system is used to show that both functions are necessary for Ofd1 to work as observed., Cells adapt to changes in ambient oxygen by changing their gene expression patterns. In fission yeast, the sterol regulatory element–binding protein Sre1 is proteolytically cleaved under low oxygen, and its N-terminal segment (Sre1N) serves as a hypoxic transcription factor. When oxygen is present, the prolyl hydroxylase Ofd1 down-regulates Sre1N activity in two ways: first, by inhibiting its binding to DNA, and second, by accelerating its degradation. Here we use a mathematical model to assess what each of these two regulatory functions contributes to the hypoxic response of the cell. By disabling individual regulatory functions in the model, which would be difficult in vivo, we found that the Ofd1 function of inhibiting Sre1N binding to DNA is essential for oxygen-dependent Sre1N regulation. The other Ofd1 function of accelerating Sre1N degradation is necessary for the yeast to quickly turn off its hypoxic response when oxygen is restored. In addition, the model predicts that increased Ofd1 production at low oxygen plays an important role in the hypoxic response, and the model indicates that the Ofd1 binding partner Nro1 tunes the response to oxygen. This model quantifies our understanding of a novel oxygen-sensing mechanism that is widely conserved.

Regulation of gene expression plays an integral role in adaptation of cells to hypoxic stress. In mammals, prolyl hydroxylases control levels of the central transcription factor hypoxia inducible factor (HIF) through regulation of HIFα subunit stability. Here, we report that the hydroxylase Ofd1 regulates the Sre1 hypoxic transcription factor in fission yeast by controlling DNA binding. Prolyl hydroxylases require oxygen as a substrate, and the activity of Ofd1 regulates Sre1-dependent transcription. In the presence of oxygen, Ofd1 binds the Sre1 N-terminal transcription factor domain (Sre1N) and inhibits Sre1-dependent transcription by blocking DNA binding. In the absence of oxygen, the inhibitor Nro1 binds Ofd1, thereby releasing Sre1N and leading to activation of genes required for hypoxic growth. In contrast to the HIF system, where proline hydroxylation is essential for regulation, Ofd1 inhibition of Sre1N does not require hydroxylation and, thus, defines a new mechanism for hypoxic gene regulation.

Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here, we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1–4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2 conjugating enzyme Ubc4, the Dsc1 RING E3 ligase and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

In fission yeast, the endoplasmic reticulum membrane-bound proteins Sre1 and Scp1, orthologs of mammalian sterol regulatory element binding protein (SREBP) and Scap, monitor sterol synthesis as an indirect measure of oxygen supply. When cellular oxygen levels are low, sterol synthesis is inhibited, and the Sre1-Scp1 complex responds by increasing transcription of genes required for adaptation to hypoxia. Sre1 and Scp1 are believed to detect a blockage in sterol synthesis by monitoring levels of particular sterols, but the evidence concerning which sterol signals this condition is unclear. Here, we demonstrate that Sre1-Scp1 senses ergosterol. Processing experimental data with a mathematical model of Sre1 and Scp1 function reveals a clear quantitative relationship between ergosterol concentration in the endoplasmic reticulum and Sre1 activation. Based on this relationship, we predict that the Sre1-Scp1 complex exists under "active" and "inactive" states and that the transition between these states is cooperatively mediated by ergosterol.

Sterol regulatory element binding proteins (SREBPs) are membrane-bound transcription factors whose proteolytic activation is controlled by the cellular sterol concentration. Mammalian SREBPs are activated in cholesterol-depleted cells and serve to regulate cellular lipid homeostasis. Recent work demonstrates that SREBP is functionally conserved in fungi. While the ability to respond to sterols is conserved, fungal SREBPs are hypoxic transcription factors required for adaptation to a low-oxygen environment. In the fission yeast Schizosaccharomyces pombe , oxygen regulates the SREBP homolog Sre1 by independently controlling both its proteolytic activation and its degradation. SREBP is also required for adaptation to hypoxia in the human pathogens Cryptococcus neoformans and Aspergillus fumigatus . In these organisms, SREBP is required for virulence and resistance to antifungal drugs, making the SREBP pathway a potential target for antifungal therapy.

In the human fungal pathogen Cryptococcus neoformans, the SREBP orthologue Sre1 is important for adaptation and growth in nutrient-limiting host tissues. In this study, we characterize the C. neoformans serotype A Sre1 and its activating protease, Stp1. We demonstrate that Stp1 is a functionally conserved orthologue of the mammalian Site-2 protease and that Stp1 cleaves Sre1 within its predicted first transmembrane segment. Gene expression analysis revealed that Stp1 is required for both Sre1-dependent and Sre1-independent gene transcription, indicating that other substrates of Stp1 may exist. Using gas chromatography, we showed that Sre1 and Stp1 are required for both normoxic and hypoxic ergosterol biosynthesis, and therefore cells lacking SRE1 or STP1 are defective for growth in the presence of low levels of the ergosterol biosynthesis inhibitors, itraconazole and 25-thialanosterol. Importantly, our studies demonstrated fungicidal effects of itraconazole and 25-thialanosterol towards sre1Delta and stp1Delta cells, demonstrating that the Sre1 pathway is required for both growth and survival in the presence of sterol biosynthesis-inhibiting antifungal drugs. Given the need for fungicidal drugs, we propose that inhibitors of Stp1, Sre1, or other regulators of Sre1 function administered in combination with a sterol synthesis inhibitor could prove an effective anticryptococcal therapy.

The endoplasmic reticulum membrane protein SREBP cleavage-activating protein (Scap) senses sterols and regulates activation of sterol-regulatory element binding proteins (SREBPs), membrane-bound transcription factors that control lipid homeostasis in fission yeast and mammals. Transmembrane segments 2–6 of Scap function as a sterol-sensing domain (SSD) that recognizes changes in cellular sterols and facilitates activation of SREBP. Previous studies identified conserved mutations Y298C, L315F, and D443N in the SSD of mammalian Scap and fission yeast Scap (Scp1) that render cells insensitive to sterols and cause constitutive SREBP activation. In this study, we utilized fission yeast genetics to identify additional functionally important residues in the SSD of Scp1 and Scap. Using a site-directed mutagenesis selection, we sampled all possible amino acid substitutions at 50 conserved residues in the SSD of Scp1 for their effects on yeast SREBP (Sre1) activation. We found mutations at 23 different amino acids in Scp1 that rendered Scp1 insensitive to sterols and caused constitutive activation of Sre1. To our surprise, the majority of the homologous Scap mutants displayed wild-type function, and only one mutation, V439G, caused constitutive activation of SREBP in mammals. These results suggest that the sterol-sensing mechanism of Scap and the functional requirements for SREBP activation are different between fission yeast and mammals.

Eukaryotic cells respond to changes in environmental oxygen supply by increasing transcription and subsequent translation of gene products required for adaptation to low oxygen. In fission yeast, the ortholog of mammalian sterol regulatory element binding protein (SREBP), called Sre1, activates low-oxygen gene expression and is essential for anaerobic growth. Previous studies in multiple organisms indicate that SREBP transcription factors function as positive regulators of gene expression by increasing transcription. Here, we describe a unique mechanism by which activation of Sre1-dependent transcription downregulates protein expression under low oxygen. Paradoxically, Sre1 inhibits expression of tco1(+) gene product by activating its transcription. Under low oxygen, Sre1 directs transcription of tco1(+) from an alternate, upstream promoter and inhibits expression of the normoxic tco1(+) transcript. The resulting low-oxygen transcript contains an additional 751 nt in the 5' untranslated region that is predicted to form a stable, complex secondary structure. Interestingly, polysome profile experiments revealed that this new longer transcript is translationally silent, leading to a decrease in Tco1 protein expression under low oxygen. Together, these results describe a new mechanism for oxygen-dependent control of gene expression and provide an example of negative regulation of protein expression by an SREBP homolog.

Hypoxic growth of fungi requires sterol regulatory element‐binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane‐anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a rhomboid family protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi‐localized protease that cleaves the transmembrane segment of the TatA rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA‐ATPase Cdc48 through a C‐terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48‐binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation. ![][1] The sterol‐responsive transcriptional regulator SREBP is proteolytically activated by sequential action of the Dsc ubiquitin ligase and Rbd2 rhomboid protease in the Golgi, representing a novel control point in the fission yeast SREBP pathway. [1]: /embed/graphic-1.gif

Cholesterol is an essential component of mammalian cell membranes and is required for proper membrane permeability, fluidity, organelle identity, and protein function. Cells maintain sterol homeostasis by multiple feedback controls that act through transcriptional and posttranscriptional mechanisms. The membrane-bound transcription factor sterol regulatory element binding protein (SREBP) is the principal regulator of both sterol synthesis and uptake. In mammalian cells, the ER membrane protein Insig has emerged as a key component of homeostatic regulation by controlling both the activity of SREBP and the sterol-dependent degradation of the biosynthetic enzyme HMG-CoA reductase. In this review, we focus on recent advances in our understanding of the molecular mechanisms of the regulation of sterol synthesis. A comparative analysis of SREBP and HMG-CoA reductase regulation in mammals, yeast, and flies points toward an equilibrium model for how lipid signals regulate the activity of sterol-sensing proteins and their downstream effectors.

In fission yeast, orthologs of mammalian SREBP and Scap, called Sre1 and Scp1, monitor oxygen-dependent sterol synthesis as a measure of cellular oxygen supply. Under low oxygen conditions, sterol synthesis is inhibited, and Sre1 cleavage is activated. However, the sterol signal for Sre1 activation is unknown. In this study, we characterized the sterol signal for Sre1 activation using a combination of Sre1 cleavage assays and gas chromatography sterol analysis. We find that Sre1 activation is regulated by levels of the 4-methyl sterols 24-methylene lanosterol and 4,4-dimethylfecosterol under conditions of low oxygen and cell stress. Both increases and decreases in the level of these ergosterol pathway intermediates induce Sre1 proteolysis in a Scp1-dependent manner. The SREBP ortholog in the pathogenic fungus Cryptococcus neoformans is also activated by high levels of 4-methyl sterols, suggesting that this signal for SREBP activation is conserved among unicellular eukaryotes. Finally, we provide evidence that the sterol-sensing domain of Scp1 is important for regulating Sre1 proteolysis. The conserved mutations Y247C, L264F, and D392N in Scp1 that render Scap insensitive to sterols cause constitutive Sre1 activation. These findings indicate that unlike Scap, fission yeast Scp1 responds to 4-methyl sterols and thus shares properties with mammalian HMG-CoA reductase, a sterol-sensing domain protein whose degradation is regulated by the 4-methyl sterol lanosterol.

Summary We investigated the effects of the hypoxia-mimetic CoCl2 in the pathogenic fungus Cryptococcus neo- formans and demonstrated that CoCl2 leads to defects in several enzymatic steps in ergosterol bio- synthesis. Sterol defects were amplified in cells lacking components of the Sre1p-mediated oxygen- sensing pathway. Consequently, Sre1p and its binding partner Scp1p were essential for growth in the presence of CoCl2. Interestingly, high copies of a single gene involved in ergosterol biosynthesis, ERG25, rescued this growth defect. We show that the inhibitory effect of CoCl2 on scp1D and sre1D cells likely resulted from either an accumulation of non-viable methylated sterols or a decrease in the amount of ergosterol. Similar findings were also observed in the ascomycetous yeast, Schizosaccha- romyces pombe, suggesting that the effects of CoCl2 on the Sre1p-mediated response are conserved in fungi. In addition, gene expression analysis revealed limited overlap between Sre1p-dependant gene activation in the presence of CoCl2 and low oxygen. The majority of genes similarly affected by both CoCl2 and low oxygen were involved in ergosterol synthesis and in iron/copper transport. This article identifies the Sre1p pathway as a common mecha- nism by which yeast cells sense and adapt to changes in both CoCl2 concentrations and oxygen levels.

Summary Cryptococcus neoformans is an environmental pathogen requiring atmospheric levels of oxygen for optimal growth. Upon inhalation, C. neoformans disseminates to the brain and causes meningoencephalitis, but the mechanisms by which the pathogen adapts to the low-oxygen environment in the brain have not been investigated. We found that SRE1, a homologue of the mammalian sterol regulatory element-binding protein (SREBP), functions in an oxygen-sensing pathway. Low oxygen decreased sterol synthesis in C. neoformans and triggered activation of membrane-bound Sre1p by the cleavage-activating protein, Scp1p. Microarray and Northern blot analysis demonstrated that under low oxygen, Sre1p activates genes required for ergosterol biosynthesis and iron uptake. Consistent with these regulatory functions, sre1Δ cells were hypersensitive to azole drugs and failed to grow under iron-limiting conditions. Importantly, sre1Δ cells failed to produce fulminating brain infection in mice. Our in vitro data support a model in which Sre1p is activated under low oxygen leading to the upregulation of genes required for sterol biosynthesis and growth in a nutrient-limiting environment. Animal studies confirm the importance of SRE1 for C. neoformans to adapt to the host environment and to cause fatal meningoencephalitis, thereby identifying the SREBP pathway as a therapeutic target for cryptococcosis.

Tumorigenesis is associated with increased glucose consumption and lipogenesis, but how these pathways are interlinked is unclear. Here, we delineate a pathway in which EGFR signaling, by increasing glucose uptake, promotes N-glycosylation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and consequent activation of SREBP-1, an endoplasmic reticulum-bound transcription factor with central roles in lipid metabolism. Glycosylation stabilizes SCAP and reduces its association with Insig-1, allowing movement of SCAP/SREBP to the Golgi and consequent proteolytic activation of SREBP. Xenograft studies reveal that blocking SCAP N-glycosylation ameliorates EGFRvIII-driven glioblastoma growth. Thus, SCAP acts as key glucose-responsive protein linking oncogenic signaling and fuel availability to SREBP-dependent lipogenesis. Targeting SCAP N-glycosylation may provide a promising means of treating malignancies and metabolic diseases.