Cholesterol and fatty acid synthesis in mammals are controlled by SREBPs, a family of membrane bound transcription factors. Our studies identified homologs of SREBP, its binding partner SCAP, and the ER retention protein Insig in Schizosaccharomyces pombe, named sre1+, scp1+, and ins1+. Like SREBP, Sre1 is cleaved and activated in response to sterol depletion in a Scp1-dependent manner. Microarray analysis revealed that Sre1 activates sterol biosynthetic enzymes as in mammals, and, surprisingly, Sre1 also stimulates transcription of genes required for adaptation to hypoxia. Furthermore, Sre1 rapidly activates these target genes in response to low oxygen and is itself required for anaerobic growth. Based on these findings, we propose and test a model in which Sre1 and Scp1 monitor oxygen-dependent sterol synthesis as an indirect measure of oxygen supply and mediate a hypoxic response in fission yeast.

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.

Using coimmunoprecipitation and tandem mass spectrometry, we identify INSIG-1 as an ER protein that binds the sterol-sensing domain of SREBP cleavage-activating protein (SCAP) and facilitates retention of the SCAP/SREBP complex in the ER. In sterol-depleted cells, SCAP escorts SREBPs from ER to Golgi for proteolytic processing, thereby allowing SREBPs to stimulate cholesterol synthesis. Sterols induce binding of SCAP to INSIG-1, as determined by blue native-PAGE, and this is correlated with the inhibition of SCAP exit from the ER. Overexpression of INSIG-1 increases the sensitivity of cells to sterol-mediated inhibition of SREBP processing. Mutant SCAP(Y298C) fails to bind INSIG-1 and is resistant to sterol-mediated inhibition of ER exit. By facilitating sterol-dependent ER retention of SCAP, INSIG-1 plays a central role in cholesterol homeostasis.

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, identification of unexpected transcriptional targets, and the discovery of functions for SREBPs in type II diabetes, cancer, immunity, neuroprotection and autophagy.

SummaryCryptococcus 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 cleavageactivating 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, sre1D cells were hypersensitive to azole drugs and failed to grow under iron-limiting conditions. Importantly, sre1D 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 nutrientlimiting 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.

The lipid composition of animal cells is controlled by SREBPs, transcription factors released from membranes by sterol-regulated proteolysis. Release is initiated by Site-1 protease (S1P), which cleaves SREBPs in the ER luminal loop between two membrane-spanning regions. To clone S1P, we prepared pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol-deprived cells, cleavage by both proteases leads to PLAP secretion. PLAP is not secreted by SRD-12B cells, cholesterol auxotrophs that lack S1P. We transfected SRD-12B cells with pCMV-PLAP-BP2 plus pools of CHO cDNAs and identified a cDNA that restores Site-1 cleavage and PLAP secretion. The cDNA encodes S1P, an intraluminal 1052-amino-acid membrane-bound subtilisin-like protease. We propose that S1P is the sterol-regulated protease that controls lipid metabolism in animal cells.

SREBPs exit the ER in a complex with SCAP. Together, they move to the Golgi where SREBP is cleaved, releasing a fragment that activates genes encoding lipid biosynthetic enzymes. Sterols block ER exit, preventing cleavage, decreasing transcription, and achieving feedback control of lipid synthesis. Here, we report an in vitro system to measure incorporation of SCAP into ER vesicles. When membranes were isolated from sterol-depleted cells, SCAP entered vesicles in a reaction requiring nucleoside triphosphates and cytosol. SCAP budding was diminished in membranes from sterol-treated cells. Kinetics of induction of budding in vitro matched kinetics of ER exit in living cells expressing GFP-SCAP. These data localize the sterol-regulated step to budding of SCAP from ER and provide a system for biochemical dissection.

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.