Ergosterol Regulates Sterol Regulatory Element Binding Protein (SREBP) Cleavage in Fission Yeast

Abstract

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. 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. IntroductionMammalian cholesterol synthesis is a tightly controlled process regulated by negative feedback inhibition (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4500) Google Scholar). The central components of this process are two endoplasmic reticulum (ER) 4The abbreviations used are: ERendoplasmic reticulumSREBPsterol regulatory element binding proteinScapSREBP cleavage activating proteinSSDsterol-sensing domain.-resident integral membrane proteins, SREBP and its binding partner Scap. SREBP is a membrane-bound transcription factor that, when proteolytically activated, enters the nucleus and induces transcription of genes required to increase intracellular cholesterol levels (2Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar). Regulated SREBP activation requires both Scap and a third ER membrane protein, Insig.Scap senses ER cholesterol concentration by directly binding cholesterol in the membrane (3Radhakrishnan A. Sun L.P. Kwon H.J. Brown M.S. Goldstein J.L. Mol. Cell. 2004; 15: 259-268Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), and cholesterol binding induces a conformational change in Scap that promotes binding to Insig, retaining the SREBP-Scap complex in the ER (4Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar, 5Sun L.P. Seemann J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6519-6526Crossref PubMed Scopus (215) Google Scholar). When cholesterol is depleted, SREBP-Scap dissociates from Insig and can then be transported to the Golgi apparatus (4Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar, 6DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The N-terminal transcription factor domain of SREBP is liberated from the membrane by two Golgi-resident proteases, allowing SREBP to enter the nucleus and activate transcription of its target genes (2Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar). Activation of SREBP target genes ultimately restores ER cholesterol, which in turn promotes Scap-Insig binding, thus blocking further ER-to-Golgi transport of SREBP-Scap and completing a negative feedback loop.Studies of SREBP in the genetically tractable fission yeast Schizosaccharomyces pombe revealed that this mammalian sterol-sensing mechanism is conserved and that SREBP functions as a principal hypoxic transcription factor in fungi (7Bien C.M. Espenshade P.J. Eukaryot. Cell. 2010; 9: 352-359Crossref PubMed Scopus (113) Google Scholar). Importantly, in the pathogenic fungi Cryptococcus neoformans and Aspergillus fumigatus, SREBP is required for virulence in fungal disease models (8Chang Y.C. Bien C.M. Lee H. Espenshade P.J. Kwon-Chung K.J. Mol. Microbiol. 2007; 64: 614-629Crossref PubMed Scopus (151) Google Scholar, 9Chun C.D. Liu O.W. Madhani H.D. PLoS Pathog. 2007; 3: e22Crossref PubMed Scopus (140) Google Scholar, 10Willger S.D. Puttikamonkul S. Kim K.H. Burritt J.B. Grahl N. Metzler L.J. Barbuch R. Bard M. Lawrence C.B. Cramer R.A. PLoS Pathog. 2008; 4: e1000200Crossref PubMed Scopus (248) Google Scholar). Fission yeast has homologs of SREBP and Scap called Sre1 and Scp1, respectively (11Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The Sre1-Scp1 complex monitors synthesis of ergosterol, the fungal equivalent of cholesterol, as an indirect measure of oxygen supply. When cellular oxygen levels are low, oxygen-dependent sterol synthesis is inhibited, and Sre1-Scp1 responds by increasing transcription of genes required for adaptation to hypoxia, including enzymes involved in ergosterol biosynthesis.Given the broad conservation, importance for fungal disease, and detailed knowledge of SREBP pathway components and function, we seek to create a mathematical model of this sterol regulatory system. As a first step, we have focused on the fission yeast Sre1 pathway. A comprehensive mathematical model would enable a thorough systems-level analysis of this pathway, including quantifying the contributions of its different parts to overall performance and evaluating the system's robustness to mutations (12Kitano H. Nat. Rev. Genet. 2004; 5: 826-837Crossref PubMed Scopus (1723) Google Scholar, 13Tomlin C.J. Axelrod J.D. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4219-4220Crossref PubMed Scopus (40) Google Scholar). Because of the conserved structure and function between the components of the mammalian and yeast SREBP pathways, such a model will likely also shed light on the process of cholesterol regulation in mammals.The Sre1 pathway can be divided conceptually into three regulatory steps as follows: Sre1 transport and cleavage, Sre1 activity in the nucleus, and ergosterol biosynthesis. As an initial step in modeling this pathway, we chose to investigate quantitatively the mechanism by which sterols control the rate of ER-Golgi transport and cleavage of Sre1-Scp1. Although mammalian cells respond to the biosynthetic end product cholesterol, our previous studies concluded that the Sre1-Scp1 complex senses 4-methyl sterols, a class of intermediates in ergosterol synthesis, as the cleavage-activating signal (14Hughes A.L. Lee C.Y. Bien C.M. Espenshade P.J. J. Biol. Chem. 2007; 282: 24388-24396Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The mechanism by which this worked was not well understood insomuch as Sre1 cleavage occurred when 4-methyl sterols both increased and decreased. Recent studies have also called into question the purity of the commercial lanosterol preparations used in these experiments (15Lange Y. Ory D.S. Ye J. Lanier M.H. Hsu F.F. Steck T.L. J. Biol. Chem. 2008; 283: 1445-1455Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).In this study, we investigated the sterol-dependent transport and cleavage of Sre1-Scp1 using a combination of experimental and mathematical techniques. We specifically sought to understand what sterol the Sre1-Scp1 complex senses, how this sensing could be described quantitatively, and what molecular mechanism could perform it. To facilitate this, we created a genetically modified strain of S. pombe that isolated the transport-cleavage process from several other factors known to affect it. Based on prior knowledge of the system, we constructed a mathematical model of this modified strain consisting of nonlinear differential equations describing the known interactions between the components in the pathway. We then performed experiments on the modified strain of yeast, interpreted the results with the aid of our model, and used the results to expand the model to include a quantitative empirical description of how the process of Sre1-Scp1 transport and cleavage responds to changes in cellular sterols. Our results suggest that ergosterol is the signal that regulates Sre1-Scp1 transport and cleavage and that the mechanism for this activation is to that used by mammalian and analysis performed as described (11Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). lanosterol and from was a of and from and and S. pombe to in yeast and of and The yeast and from by Espenshade P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. M.S. A. A. PubMed Scopus Google and Sre1 as described (11Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (260) Google cleavage was by of of oxygen in for such that cells a of the of for and different and for to protein with to the into a for (11Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). using the with The cleavage of Sre1 Sre1 was using signals from of of sterols was performed as described (14Hughes A.L. Lee C.Y. Bien C.M. Espenshade P.J. J. Biol. Chem. 2007; 282: 24388-24396Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). of and sterols, yeast cells as by for with in and to a of was to the which was then divided in and in of for with of and with of and by as described (14Hughes A.L. Lee C.Y. Bien C.M. Espenshade P.J. J. Biol. Chem. 2007; 282: 24388-24396Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). was for sterol by the between of and sterol by the of Sre1 understand the relationship between the rate of Sre1-Scp1 transport and cleavage and the of Sre1 cleavage we a mathematical model of Sre1 in yeast model a of cells and thus the to model and the model to be used with data from of the in this model, described under with rate The and Scp1, Sre1-Scp1 and Because the Sre1 feedback is in this strain under we can that Sre1 and Scp1 are by transcription and concentration and We a between Sre1 and these proteins with a rate and with a rate we transport and cleavage of Sre1-Scp1 complex as a step and that the rate for this step, is not but changes in for the of the Sre1-Scp1 complex to increase its rate of transport and cleavage likely a change in the rate of sterol We also that, as of the transport and cleavage step, Scp1 is liberated from the Sre1-Scp1 complex and to the ER to Sre1-Scp1 P.J. A.L. Rev. Genet. 2007; PubMed Scopus Google Scholar). is to the of the of this a rate to the concentration of the with a rate A mathematical for this step and a for are in the of in we can rate equations to the of this as in in Sre1 transport-cleavage mechanism in in in a The concentration of Sre1 in the is can from that the of Sre1 and to will the that and the experimental not change the of Sre1 then is the of we can the model by by the and as in we the concentration of Scp1 in the model as then and the rate of Scp1 then will the that and that the experimental not affect Scp1 then is also the of the of the model, we its rate cells have a of which Because is we can the of to In the concentration of Scp1 is to that of Sre1 and on Sre1 levels in cells of Sre1 levels under and is we the of to to We also and a rate for Sre1-Scp1 transport and cleavage, we can to the of in our we have the for the of an we to the transport-cleavage rate that to that of To this, we the and as well as a for from to we Sre1 and by quantifying as described and We then for the of the by the with a in C.B. with for and Scholar). in the model we the of To an initial condition for the we used the to be the of to the and initial condition we with to for the of the we for by in the described under experimental evidence to ergosterol as the signal that regulates the transport and cleavage of the Sre1-Scp1 of the ergosterol in a yeast is not in the ER J. PubMed Scopus Google and thus is not by Sre1 and Scp1, which are in the ER We used a mathematical model to the concentration of ergosterol in the from the of ergosterol both and and for a to of and is by the structure of the model of Sre1 for the of required to a of reveals that with for and for of Because this relationship the that in Sre1 levels to in the of is to the of when a to it. when the to of and we the of between the of the and the of the by the and of the and for the of the was performed by nonlinear in the rate for Sre1-Scp1 transport and cleavage, is a function of ergosterol in the ER and not of other sterol pathway intermediates on data from experiments with different to the sterol pathway as in in the is for the of in The in A is a function with and to the between the of the and the of the the of Sre1 cleavage by model with from experimental we an that into how in the the of Sre1 cleavage is a of to a in but the is the of to a in Because of this, our described in in the how in the and would be required to a in with of in experiments used to and the and light and of using the described under is the between the of and the by the data as described under this study, we that ergosterol regulates the rate of transport and cleavage of the Sre1-Scp1 complex in fission experiments in which of lanosterol Sre1 cleavage to be as the lanosterol used a that ergosterol and the concentration of cellular ergosterol to further which concluded that ergosterol is the signal Sre1-Scp1 transport and of evidence this different of ergosterol synthesis, which steps both and in the ergosterol biosynthetic pathway, Sre1 cleavage cleavage is with a in ergosterol concentration but with changes in the concentration of other sterol pathway intermediates levels of ergosterol by blocking ergosterol Sre1 cleavage the transport-cleavage rate is a function of ergosterol but not other sterol pathway intermediates Importantly, the mathematical model used to from experimental data as to the of the signal the model using ER ergosterol as the signal the of the Because ergosterol is the end product of the sterol biosynthetic pathway in yeast and the sterol of the yeast membrane and J. PubMed Scopus Google Scholar), is that the Sre1-Scp1 complex monitors ergosterol to the of sterol sensing of ergosterol by Sre1-Scp1 is of between the sterol regulatory in S. pombe and that in as SREBP and Scap monitor levels of cholesterol, the end product of mammalian sterol synthesis P.J. A.L. Rev. Genet. 2007; PubMed Scopus Google Scholar). Scap an in which three and are for the SREBP-Scap complex to Insig and be in the mutations of these the complex D. C.M. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: PubMed Scopus Google Scholar). Although Scp1 and Scap have these three are conserved in Scp1 and with of the protein these (11Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). mutations of these three in Scp1 to increased Sre1-Scp1 cleavage changes in the sterol (14Hughes A.L. Lee C.Y. Bien C.M. Espenshade P.J. J. Biol. Chem. 2007; 282: 24388-24396Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). that the in yeast Scp1 functions to that of mammalian between Scp1 sensing ergosterol in fission yeast and Scap sensing cholesterol in a molecular mechanism for how ergosterol regulates ER-Golgi transport of Sre1-Scp1 and cleavage of Scap cholesterol levels by binding directly to cholesterol (3Radhakrishnan A. Sun L.P. Kwon H.J. Brown M.S. Goldstein J.L. Mol. Cell. 2004; 15: 259-268Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), and cholesterol changes the of Scap C.M. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, Sun J.D. Brown M.S. Goldstein J.L. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). promotes Scap binding to Insig, which the SREBP-Scap complex in the SREBP cleavage in the Golgi (4Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar, 5Sun L.P. Seemann J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6519-6526Crossref PubMed Scopus (215) Google Scholar). cholesterol has to SREBP-Scap cleavage in the of Insig L.P. Seemann J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6519-6526Crossref PubMed Scopus (215) Google Scholar). has that cholesterol regulates SREBP-Scap transport in a A. Goldstein J.L. Brown M.S. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). data suggest a model in which Scap exists in two as follows: “active” that is for transport and cleavage, and “inactive” that is in the ER and to Insig P.J. A.L. Rev. Genet. 2007; PubMed Scopus Google Scholar). In this model, cholesterol cooperatively promotes the transition from the to the on our results we a model for regulation of Sre1-Scp1 cleavage by ergosterol, that the fission yeast of Insig, not a in this regulation (11Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, R. A.L. Espenshade P.J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). that Scp1, Scap, has two an and an in and the transition between these is cooperatively on the concentration of ergosterol in the ER with a for and we have pathway in yeast with a mechanism for regulation of Sre1-Scp1 transport and cleavage by ergosterol. Scp1 to Sre1 has two an and an The between these is and cooperatively on levels of ergosterol in the The is transported from the ER to the Golgi and to with a rate our model of Sre1-Scp1 transport described is the rate for Sre1-Scp1 transport and cleavage, the rate of transport and cleavage of concentration of that the transport-cleavage rate is a but the is for the rate of transport and cleavage is and the concentration of Sre1-Scp1 complex is the of we have under this function our that a mechanism ergosterol in the ER cooperatively a transition between and of Sre1-Scp1 complex is a for our studies will be required to the of of Scp1 and the of ergosterol in the model Sre1-Scp1 transport and cleavage with a of quantitative the of our of Sre1 are to experimental and the of our for to this this model not include the process of ergosterol which the of ergosterol to be by the Sre1-Scp1 with these the model of the in the data that the of the Sre1-Scp1 transport-cleavage the process of the for ergosterol sensing by Sre1-Scp1 a Although this model was in a genetically modified strain of S. be to yeast, as we have to that in this strain directly the that Sre1-Scp1 transport and this study, we evidence that ergosterol regulates the ER-Golgi transport and cleavage of the fission yeast homologs of mammalian we a quantitative empirical model that the of this regulation and a molecular mechanism by which could The experiments used to and this model in a modified yeast strain that of the in the Sre1-Scp1 of we this model to as a that will be in the with models of the other ultimately allowing of a model for the fission yeast SREBP pathway. IntroductionMammalian cholesterol synthesis is a tightly controlled process regulated by negative feedback inhibition (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4500) Google Scholar). The central components of this process are two endoplasmic reticulum (ER) 4The abbreviations used are: ERendoplasmic reticulumSREBPsterol regulatory element binding proteinScapSREBP cleavage activating proteinSSDsterol-sensing domain.-resident integral membrane proteins, SREBP and its binding partner Scap. SREBP is a membrane-bound transcription factor that, when proteolytically activated, enters the nucleus and induces transcription of genes required to increase intracellular cholesterol levels (2Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar). Regulated SREBP activation requires both Scap and a third ER membrane protein, Insig.Scap senses ER cholesterol concentration by directly binding cholesterol in the membrane (3Radhakrishnan A. Sun L.P. Kwon H.J. Brown M.S. Goldstein J.L. Mol. Cell. 2004; 15: 259-268Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), and cholesterol binding induces a conformational change in Scap that promotes binding to Insig, retaining the SREBP-Scap complex in the ER (4Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar, 5Sun L.P. Seemann J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6519-6526Crossref PubMed Scopus (215) Google Scholar). When cholesterol is depleted, SREBP-Scap dissociates from Insig and can then be transported to the Golgi apparatus (4Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar, 6DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The N-terminal transcription factor domain of SREBP is liberated from the membrane by two Golgi-resident proteases, allowing SREBP to enter the nucleus and activate transcription of its target genes (2Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar). Activation of SREBP target genes ultimately restores ER cholesterol, which in turn promotes Scap-Insig binding, thus blocking further ER-to-Golgi transport of SREBP-Scap and completing a negative feedback loop.Studies of SREBP in the genetically tractable fission yeast Schizosaccharomyces pombe revealed that this mammalian sterol-sensing mechanism is conserved and that SREBP functions as a principal hypoxic transcription factor in fungi (7Bien C.M. Espenshade P.J. Eukaryot. Cell. 2010; 9: 352-359Crossref PubMed Scopus (113) Google Scholar). Importantly, in the pathogenic fungi Cryptococcus neoformans and Aspergillus fumigatus, SREBP is required for virulence in fungal disease models (8Chang Y.C. Bien C.M. Lee H. Espenshade P.J. Kwon-Chung K.J. Mol. Microbiol. 2007; 64: 614-629Crossref PubMed Scopus (151) Google Scholar, 9Chun C.D. Liu O.W. Madhani H.D. PLoS Pathog. 2007; 3: e22Crossref PubMed Scopus (140) Google Scholar, 10Willger S.D. Puttikamonkul S. Kim K.H. Burritt J.B. Grahl N. Metzler L.J. Barbuch R. Bard M. Lawrence C.B. Cramer R.A. PLoS Pathog. 2008; 4: e1000200Crossref PubMed Scopus (248) Google Scholar). Fission yeast has homologs of SREBP and Scap called Sre1 and Scp1, respectively (11Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The Sre1-Scp1 complex monitors synthesis of ergosterol, the fungal equivalent of cholesterol, as an indirect measure of oxygen supply. When cellular oxygen levels are low, oxygen-dependent sterol synthesis is inhibited, and Sre1-Scp1 responds by increasing transcription of genes required for adaptation to hypoxia, including enzymes involved in ergosterol biosynthesis.Given the broad conservation, importance for fungal disease, and detailed knowledge of SREBP pathway components and function, we seek to create a mathematical model of this sterol regulatory system. As a first step, we have focused on the fission yeast Sre1 pathway. A comprehensive mathematical model would enable a thorough systems-level analysis of this pathway, including quantifying the contributions of its different parts to overall performance and evaluating the system's robustness to mutations (12Kitano H. Nat. Rev. Genet. 2004; 5: 826-837Crossref PubMed Scopus (1723) Google Scholar, 13Tomlin C.J. Axelrod J.D. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4219-4220Crossref PubMed Scopus (40) Google Scholar). Because of the conserved structure and function between the components of the mammalian and yeast SREBP pathways, such a model will likely also shed light on the process of cholesterol regulation in mammals.The Sre1 pathway can be divided conceptually into three regulatory steps as follows: Sre1 transport and cleavage, Sre1 activity in the nucleus, and ergosterol biosynthesis. As an initial step in modeling this pathway, we chose to investigate quantitatively the mechanism by which sterols control the rate of ER-Golgi transport and cleavage of Sre1-Scp1. Although mammalian cells respond to the biosynthetic end product cholesterol, our previous studies concluded that the Sre1-Scp1 complex senses 4-methyl sterols, a class of intermediates in ergosterol synthesis, as the cleavage-activating signal (14Hughes A.L. Lee C.Y. Bien C.M. Espenshade P.J. J. Biol. Chem. 2007; 282: 24388-24396Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The mechanism by which this worked was not well understood insomuch as Sre1 cleavage occurred when 4-methyl sterols both increased and decreased. Recent studies have also called into question the purity of the commercial lanosterol preparations used in these experiments (15Lange Y. Ory D.S. Ye J. Lanier M.H. Hsu F.F. Steck T.L. J. Biol. Chem. 2008; 283: 1445-1455Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).In this study, we investigated the sterol-dependent transport and cleavage of Sre1-Scp1 using a combination of experimental and mathematical techniques. We specifically sought to understand what sterol the Sre1-Scp1 complex senses, how this sensing could be described quantitatively, and what molecular mechanism could perform it. To facilitate this, we created a genetically modified strain of S. pombe that isolated the transport-cleavage process from several other factors known to affect it. Based on prior knowledge of the system, we constructed a mathematical model of this modified strain consisting of nonlinear differential equations describing the known interactions between the components in the pathway. We then performed experiments on the modified strain of yeast, interpreted the results with the aid of our model, and used the results to expand the model to include a quantitative empirical description of how the process of Sre1-Scp1 transport and cleavage responds to changes in cellular sterols. Our results suggest that ergosterol is the signal that regulates Sre1-Scp1 transport and cleavage and that the mechanism for this activation is to that used by mammalian Scap.