Degradation of Sterol Regulatory Element-binding Protein Precursor Requires the Endoplasmic Reticulum-associated Degradation Components Ubc7 and Hrd1 in Fission Yeast

Abstract

Sre1, the fission yeast sterol regulatory element-binding protein (SREBP), is an endoplasmic reticulum (ER) membrane-bound transcription factor that is a principal regulator of hypoxic gene expression. Under low oxygen, Sre1 is cleaved from its inactive ER precursor form to generate an active nuclear transcription factor that up-regulates genes required for low oxygen growth. To maintain a constant supply of Sre1, Sre1 precursor synthesis must be regulated to replenish Sre1 precursor lost to proteolytic cleavage under low oxygen. In this study, we investigated the mechanisms controlling Sre1 precursor levels. We found that positive feedback regulation at the sre1+ promoter increases the synthesis of the Sre1 precursor under low oxygen and that this regulation is required for maximal Sre1 activation and target gene expression. We also demonstrate that the Sre1 precursor is rapidly degraded by the proteasome in the absence of its binding partner Scp1, which is required for oxygen-regulated Sre1 cleavage. Degradation of Sre1 in the absence of Scp1 requires the ER-associated degradation (ERAD) components Ubc7, an E2 ubiquitin conjugating enzyme, and Hrd1, an E3 ubiquitin ligase. We conclude that positive feedback regulation to up-regulate Sre1 precursor synthesis under low oxygen is essential for Sre1 function and propose that excess Sre1 precursor is removed by ERAD to ensure complex formation between Sre1 and its binding partner Scp1. Thus, Sre1 is a new example of an endogenous ERAD substrate, establishing fission yeast as an organism for the study of this important degradative pathway. Sre1, the fission yeast sterol regulatory element-binding protein (SREBP), is an endoplasmic reticulum (ER) membrane-bound transcription factor that is a principal regulator of hypoxic gene expression. Under low oxygen, Sre1 is cleaved from its inactive ER precursor form to generate an active nuclear transcription factor that up-regulates genes required for low oxygen growth. To maintain a constant supply of Sre1, Sre1 precursor synthesis must be regulated to replenish Sre1 precursor lost to proteolytic cleavage under low oxygen. In this study, we investigated the mechanisms controlling Sre1 precursor levels. We found that positive feedback regulation at the sre1+ promoter increases the synthesis of the Sre1 precursor under low oxygen and that this regulation is required for maximal Sre1 activation and target gene expression. We also demonstrate that the Sre1 precursor is rapidly degraded by the proteasome in the absence of its binding partner Scp1, which is required for oxygen-regulated Sre1 cleavage. Degradation of Sre1 in the absence of Scp1 requires the ER-associated degradation (ERAD) components Ubc7, an E2 ubiquitin conjugating enzyme, and Hrd1, an E3 ubiquitin ligase. We conclude that positive feedback regulation to up-regulate Sre1 precursor synthesis under low oxygen is essential for Sre1 function and propose that excess Sre1 precursor is removed by ERAD to ensure complex formation between Sre1 and its binding partner Scp1. Thus, Sre1 is a new example of an endogenous ERAD substrate, establishing fission yeast as an organism for the study of this important degradative pathway. Activation of low oxygen gene expression in Schizosaccharomyces pombe is primarily controlled by the Sre1 transcription factor. Sre1 is the fission yeast homolog of the mammalian sterol regulatory element-binding protein (SREBP) 4The abbreviations used are: SREBPsterol regulatory element-binding proteinScapSREBP cleavage activating proteinERendoplasmic reticulumERADendoplasmic reticulum-associated degradationSRESre1 regulatory elementYESyeast extract plus supplementsCaMVcauliflower mosaic virusMPmutant promoterHMG-CoA3-hydroxy-3-methylglutaryl-coenzyme A. (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). SREBPs are ER-membrane bound, basic helix-loop-helix transcription factors that control expression of genes required for uptake and synthesis of cholesterol under sterol limiting conditions (2Espenshade P.J. Hughes A.L. Annu. Rev. Genet. 2007; 41: 401-427Crossref PubMed Scopus (439) Google Scholar). SREBP forms a tight complex with its binding partner Scap (SREBP cleavage activating protein). When sterol levels are low, Scap escorts SREBP to the Golgi where SREBP is sequentially cleaved by Site 1 and Site 2 proteases to release the N-terminal transcription factor domain (3Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1291) Google Scholar). Sterols regulate access of SREBP to the Golgi-localized proteases by controlling entrance of SREBP-Scap into ER transport vesicles (4Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Following SREBP cleavage in the Golgi, Scap is thought to recycle to the ER and bind to newly synthesized SREBP to participate in additional rounds of cleavage (2Espenshade P.J. Hughes A.L. Annu. Rev. Genet. 2007; 41: 401-427Crossref PubMed Scopus (439) Google Scholar). sterol regulatory element-binding protein SREBP cleavage activating protein endoplasmic reticulum endoplasmic reticulum-associated degradation Sre1 regulatory element yeast extract plus supplements cauliflower mosaic virus mutant promoter 3-hydroxy-3-methylglutaryl-coenzyme A. In fission yeast, Sre1 is proteolytically cleaved and activated in response to low oxygen (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Under low oxygen conditions, oxygen-dependent ergosterol synthesis is inhibited, leading to Sre1 cleavage and release of the N-terminal transcription factor domain (Sre1N). In addition, oxygen also controls the degradation of Sre1N. In the presence of oxygen, the prolyl hydroxylase family member Ofd1 accelerates the turnover of Sre1N (5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar). In the absence of oxygen, Ofd1 is inhibited leading to rapid accumulation of cleaved Sre1N and expression of Sre1 target genes. Sre1 activates expression of genes in pathways involved in non-respiratory oxygen consumption required for low oxygen growth (6Todd B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). Like mammalian SREBP, activation of Sre1 also requires binding to Scap, called Scp1 in fission yeast (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Scap contains a sterol sensing domain in transmembrane domains 2–6 that is required for sterol-regulated cleavage of SREBPs (2Espenshade P.J. Hughes A.L. Annu. Rev. Genet. 2007; 41: 401-427Crossref PubMed Scopus (439) Google Scholar). Interestingly, in both yeast and mammals, the SREBP precursor is reduced in the absence of Scap (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 7Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The mechanism by which the SREBP precursor is regulated in the absence of Scap is unknown. Both luminal and membrane-bound ER proteins are subject to quality control and misfolded proteins are degraded by a process termed ER-associated protein degradation (ERAD) (8Ellgaard L. Helenius A. Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1706) Google Scholar). ERAD involves recognition and polyubiquitination of misfolded protein substrates, extraction from the ER, and degradation by the proteasome (9Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (407) Google Scholar, 10Meusser B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (1022) Google Scholar, 11Sayeed A. Ng D.T. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 75-91Crossref PubMed Scopus (51) Google Scholar). In Saccharomyces cerevisiae, the ERAD pathway involves two ubiquitin ligases, Hrd1p and Doa10p, both of which contain cytosolic RING finger domains (12Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (237) Google Scholar). Hrd1p is required for degradation of protein substrates with misfolded ER-luminal or membrane domains and Doa10p is required for degradation of proteins with misfolded cytosolic domains (13Carvalho P. Goder V. Rapoport T.A. Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 14Huyer G. Piluek W.F. Fansler Z. Kreft S.G. Hochstrasser M. Brodsky J.L. Michaelis S. J. Biol. Chem. 2004; 279: 38369-38378Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Both the Hrd1p and Doa10p E3 ubiquitin ligases can catalyze the polyubiquitination of substrates through the ubiquitin-conjugating enzyme Ubc7p (15Kostova Z. Tsai Y.C. Weissman A.M. Semin. Cell Dev. Biol. 2007; 18: 770-779Crossref PubMed Scopus (132) Google Scholar). In the sterol pathway, S. cerevisiae Hrd1 and the mammalian E3 ligase gp78 catalyze the ubiquitination and degradation of the sterol biosynthetic enzyme HMG-CoA reductase in sterol-overloaded cells (9Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (407) Google Scholar, 16Song B.L. Javitt N.B. DeBose-Boyd R.A. Cell Metab. 2005; 1: 179-189Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In addition, gp78 mediates sterol-regulated degradation of Insig-1, a central regulator of cholesterol homeostasis (17Lee J.N. Song B. DeBose-Boyd R.A. Ye J. J. Biol. Chem. 2006; 281: 39308-39315Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Therefore, ERAD functions in the physiological regulated degradation of proteins as well as degradation of misfolded proteins. Our previous studies revealed that oxygen controls Sre1 proteolytic activation and nuclear Sre1 protein stability to regulate Sre1-dependent target gene expression (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar). However, to achieve sustained activation of Sre1 target genes under low oxygen, cleaved Sre1N must be continually produced from the Sre1 precursor. Here, we investigated the mechanisms regulating Sre1 precursor levels independent of oxygen-regulated proteolytic cleavage. We found that positive feedback regulation of the sre1+ promoter is necessary to maintain Sre1 precursor levels and is required for maximal Sre1 activation and function. We also demonstrate that Sre1 precursor levels are dependent upon Scp1. In the absence of Scp1, the Sre1 precursor is rapidly degraded by the proteasome in a mechanism requiring the ERAD components, Ubc7 and Hrd1. These results demonstrate that positive feedback is required for Sre1 pathway function and suggest that Sre1 is an endogenous substrate for ER-associated degradation. Yeast extract from from and from from and from proteasome from S. pombe and from the Mol. Genet. PubMed Scopus Google Scholar). S. pombe and from yeast or by J. M.S. A. A. P. 14: PubMed Scopus Google Scholar). The and (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). at the by in yeast Mol. Genet. PubMed Scopus Google Scholar). The to The a from mutant contains Sre1 binding and in the sre1+ promoter to positive feedback regulation by Sre1 (6Todd B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). The of from to and the of from to To generate to of the sre1+ with by with a of the sre1+ and and the (6Todd B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). to for expression of the gene J. G. PubMed Scopus Google Scholar). by and by used to generate the and S. A. P. PubMed Scopus Google Scholar). Yeast to at in yeast extract plus supplements of and or in where (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). growth conditions an In as (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). yeast or and for which sre1+ from the cauliflower mosaic virus promoter in sre1+ from S. pombe and into and to this with and the sre1+ into the of to (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). with and the sre1+ into to PubMed Scopus Google Scholar). and as well as for and (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar). yeast extract membrane protein and and (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, A.L. Stewart E.V. Espenshade P.J. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). protein the yeast in the presence or absence of oxygen to where and to conditions in the presence of and to the or the to and the results of at independent Yeast cells with in and and with and at with and and to The of Sre1 precursor is controlled by of oxygen-regulated proteolytic and degradation. we that Sre1 to its promoter and up-regulates expression of sre1+ in a positive feedback mechanism (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar, B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). To the of this positive feedback to Sre1 precursor levels and of the Sre1 pathway, we two Sre1 binding and in the sre1+ promoter required for positive feedback regulation and this (5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar, B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). To in and the low oxygen in sre1+ we and cells in the presence or absence of oxygen for cells in the absence of oxygen a in Sre1N and a in the sre1+ 1 and In sre1+ in cells in the absence of oxygen and Sre1 cleavage reduced and expression of a Sre1 target also reduced in cells with cells in the absence of oxygen (6Todd B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). sre1+ and Sre1 precursor levels in and cells in the presence of oxygen, that the and required for synthesis of the sre1+ or Sre1 precursor in conditions 1 and Sre1 precursor reduced in cells in the absence of oxygen 2 and that sre1+ expression under low oxygen is required to maintain a supply of Sre1 precursor. To the of Sre1 precursor in and cells in the absence of oxygen for in cells Sre1 cleavage maximal at and Sre1 precursor under low oxygen (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). However, in cells Sre1 precursor and by of low oxygen growth accumulation of Sre1N maximal at 2 and at and the of Sre1 precursor results that positive feedback regulation of the sre1+ promoter is required for maximal Sre1 activation and target gene expression under low oxygen. Our previous studies that Sre1 cleavage is activated by a that sterol synthesis Hughes A.L. Espenshade P.J. Y.C. Mol. 2007; PubMed Scopus Google and that sre1+ is required for growth To positive feedback regulation at the sre1+ promoter is required for Sre1 we growth of or cells cells growth to Hughes A.L. Espenshade P.J. Y.C. Mol. 2007; PubMed Scopus Google Scholar). cells to cells However, cells a growth with reduced Sre1 function results demonstrate that positive feedback regulation of sre1+ is required for maximal Sre1 activation and Sre1 pathway function. that positive feedback regulation is required to maintain the Sre1 precursor we investigated Sre1 precursor degradation is cells in the presence of oxygen to Sre1 proteolytic cleavage. In the ER, Sre1 forms a complex with Scp1 (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). In the absence of Scp1, Sre1 precursor reduced 1 and To the in Sre1 precursor to Sre1 we a to Sre1 in and and cells with to protein and In Sre1 precursor However, in cells Sre1 degradation These results that Scp1 is required for Sre1 precursor stability in to its in Sre1 proteolytic cleavage. Interestingly, of Sre1 precursor 2 and that excess Scp1 additional levels of To Sre1 in controls the stability of Scp1, we Scp1 in and cells We in Scp1 levels and revealed that Scp1 is a protein is dependent upon Sre1 results demonstrate that Sre1 precursor protein stability is dependent Scp1 and that Scp1 is a protein under Sre1 is in the absence of Scp1, we to the mechanism by which Sre1 is To Sre1 degradation in cells requires the and cells with a proteasome for with the proteasome Sre1 precursor levels in cells and Interestingly, with proteasome also the Sre1 precursor in cells 1 and that Sre1 precursor is subject to regulation in To the in Sre1 precursor in cells is to protein we a cells with or proteasome for with proteasome the stability of the Sre1 precursor with the control that of the proteasome increases Sre1 To the proteasome is required for Sre1 we the of Sre1 precursor in cells that a in a regulatory of the proteasome C. G. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and cells for at the and for cells Sre1 precursor cells and that at the the proteasome inhibited in cells and Sre1 at Sre1 precursor in in cells that of the proteasome Sre1 precursor in the absence of Scp1. In addition, growth of cells also the Sre1 precursor that the Sre1 precursor in cells results demonstrate that degradation of the Sre1 precursor requires the the that Sre1 is an ER-membrane its stability in the absence of Scp1 and its turnover required the proteasome we that components of the ERAD pathway be required for Sre1 degradation. Interestingly, the S. pombe homolog of the S. cerevisiae which for an E2 ubiquitin-conjugating enzyme, as a Sre1-dependent target gene by (6Todd B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar, G. Cell. 2002; 1: PubMed Scopus Google Scholar). Sre1 target genes regulate Sre1, we investigated Ubc7 is required for Sre1 degradation (5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar). We a and Sre1 precursor by In cells the Sre1 precursor of the Sre1 precursor 2 and of Sre1 precursor in and Sre1N in the absence of oxygen that Scp1 is required for Sre1 cleavage in to Sre1 stability and levels of Sre1 cleavage in the absence of oxygen and To the in Sre1 precursor in cells to protein we a and of the of the Sre1 precursor in that Ubc7 is required for rapid degradation of Sre1 in the absence of Scp1 To the of Sre1 to the precursor form of Sre1, we the of Sre1N in and To the of cells in the absence of oxygen for to generate Sre1N by proteolytic cleavage. to the presence of oxygen to Sre1 precursor and Sre1N a of turnover in both and cells that the function of Ubc7 in Sre1 degradation is for the Sre1 precursor. Scp1 levels in and that Scp1 is for degradation by the pathway 1 and results demonstrate that the E2 ubiquitin-conjugating enzyme Ubc7 is required for Sre1 precursor degradation. E3 ubiquitin ligases, and Hrd1, catalyze polyubiquitination of ER protein substrates through Ubc7 in S. cerevisiae (15Kostova Z. Tsai Y.C. Weissman A.M. Semin. Cell Dev. Biol. 2007; 18: 770-779Crossref PubMed Scopus (132) Google Scholar). of and in S. pombe R. M. Hochstrasser M. Dev. PubMed Scopus Google Scholar). To or Hrd1 are required for Sre1 degradation in we and yeast and the Sre1 precursor. of the Sre1 precursor in of and with the results in of Sre1 precursor in cells the of Scp1 for Sre1 cleavage under low oxygen and To Hrd1 is required for stability of Sre1 in Sre1 degradation in a and Sre1 rapidly degraded in cells and of Sre1 Hrd1 functions to the Sre1 precursor as of the degradation of Sre1N and cells levels of Scp1 with cells that Hrd1 and control Scp1 levels E3 ubiquitin ligases to the by binding and substrate proteins for degradation B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (1022) Google Scholar). To Hrd1 forms a complex with the Sre1 we an in In the absence of Scp1, Sre1 precursor be for ERAD by Hrd1, ubiquitination and degradation be to of from a complex with the Sre1 the ER membrane protein HMG-CoA and results that the E3 ubiquitin ligase Hrd1 is required to the Sre1 precursor in the absence of Scp1. Sre1 precursor supply is controlled by new synthesis and degradation. To the of the degradative pathway to Sre1 we degradation of the Sre1 precursor in Sre1 cleavage in cells positive feedback regulation of To we a to positive feedback regulation and degradation of the Sre1 precursor. We and cells in the presence and absence of oxygen for and for and Under low oxygen, cells Sre1 cleaved Sre1, and with cells Interestingly, of in cells Sre1 cleavage or under low oxygen conditions with of growth of cells Thus, of Sre1 precursor degradation in Sre1 cleavage and is to the for positive feedback regulation of Our that Ubc7 and Hrd1 function to the Sre1 precursor that is to Scp1 we that the degradative pathway functions to Sre1 produced in excess of Scp1. To the of Ubc7 and Hrd1 to Sre1 precursor degradation Scp1 is we a in and cells in the presence of oxygen. Ubc7 or Hrd1 Sre1 precursor at However, the of the Sre1 precursor in that a of Sre1 precursor is degraded by ERAD in cells in the presence of oxygen. The turnover of Sre1 precursor in and cells that Sre1 is proteolytically activated to Sre1N at a in the presence of oxygen. To that also function to Sre1 to Scp1 in we a in which Sre1 synthesis Scp1. Under low oxygen, cells up-regulate sre1+ transcription through positive feedback regulation to maintain a supply of Sre1 precursor for proteolytic cleavage When oxygen is Sre1 cleavage is (5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google the complex and the of Scp1 to bind to the newly synthesized Sre1 precursor is Thus, under this we that the of Sre1 synthesis the of Scp1. this we and cells for in the presence of oxygen, the absence of oxygen, or the absence of oxygen for and plus oxygen for 1 In Sre1 proteolytically activated under low oxygen and the Sre1 precursor 1 and to the presence of oxygen, Sre1N that Sre1 cleavage inhibited under conditions In and the Sre1 precursor in the presence of oxygen and cells levels of Sre1 precursor under low oxygen and both and cells levels of Sre1 precursor to cells to the presence of oxygen for 1 and These results that Ubc7 and Hrd1 function to the Sre1 precursor in The for Ubc7 and Hrd1 is upon the synthesis of Sre1 the of Scp1 to bind newly synthesized Under low oxygen, Sre1-dependent target gene expression increases to proteolytic cleavage of the Sre1 precursor and of nuclear Sre1N (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar). However, to achieve sustained activation of Sre1 target genes Sre1N must be continually produced from the Sre1 precursor by proteolytic cleavage. Here, we investigated the mechanisms by which the Sre1 precursor is regulated independent of oxygen-regulated proteolytic cleavage. we that Sre1 up-regulates its expression by a positive feedback mechanism (5Hughes B.T. Espenshade P.J. EMBO J. 2008; 27: 1491-1501PubMed Google Scholar, B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). a yeast in the sre1+ we found that positive feedback regulation of Sre1 synthesis is required for sustained activation of Sre1N under low oxygen and growth of cells These the of this positive feedback regulation for Sre1 pathway function. We also investigated mechanisms regulating Sre1 precursor degradation. We found that in the absence of the partner Scp1, the Sre1 precursor is rapidly degraded by the proteasome this degradation requires Ubc7 and Hrd1, components of the ERAD pathway, and the Sre1 precursor a complex with Hrd1 (12Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (237) Google Scholar). we that Sre1 is an endogenous substrate for the pathway in cells These suggest that Sre1 is a substrate for the E3 ubiquitin ligase Hrd1. However, to we to Sre1 for the degradation of Our a in which are to regulate Sre1 precursor synthesis of the Sre1 precursor is controlled by positive feedback the E2 ubiquitin-conjugating enzyme Ubc7 and the E3 ubiquitin ligase Hrd1 the degradation of Sre1 precursor to and the Sre1 precursor is subject to cleavage under low oxygen to Sre1N (1Hughes A.L. Todd B.L. Espenshade P.J. Cell. 2005; 120: 831-842Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, B.L. Stewart E.V. Burg J.S. Hughes A.L. Espenshade P.J. Mol. Cell Biol. 2006; 26: 2817-2831Crossref PubMed Scopus (141) Google Scholar). In the ER, the Sre1 precursor as Sre1 or complex Under low oxygen, Sre1 to Scp1 is proteolytically cleaved to release the N-terminal transcription factor Sre1N the and up-regulates sre1+ expression by a positive feedback mechanism that results in synthesis of the Sre1 precursor and sustained cleavage under low oxygen In this study, we also that Sre1 is in the absence of Scp1 and is degraded by the proteasome the pathway we that Sre1 is as a and is degraded by an mechanism requiring Ubc7 and Hrd1 (8Ellgaard L. Helenius A. Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1706) Google Scholar, K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (237) Google Scholar). degradative mechanisms control complex formation of the complex and the yeast (8Ellgaard L. Helenius A. Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1706) Google Scholar, S. S. EMBO 2007; PubMed Scopus Google Scholar). with Scp1, Sre1 is as misfolded and is from the rapid degradation of Sre1 that Sre1 in a complex with Scp1, that Sre1 cleavage is regulated by Scp1 and oxygen that the Sre1 precursor is a physiological substrate for ER quality studies fission yeast as a new organism for the of this important degradative pathway. Like fission yeast Sre1, mammalian SREBP precursor is also reduced in the absence of Scap, to protein stability R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). is an mechanism also controls this degradation. Interestingly, of the ER-associated ubiquitin ligase expression of the SREBP target genes HMG-CoA reductase and in in regulation of SREBP A. Tsai Y.C. D. Weissman A.M. 2007; 26: PubMed Scopus Google Scholar). studies are required to the pathways regulating SREBP protein stability in mammalian cells and the for in this We Espenshade and Michaelis for and for the We also for the