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Activation of Sterol Regulatory Element-binding Protein by the Caspase Drice in Drosophila Larvae

2009; Elsevier BV; Volume: 284; Issue: 15 Linguagem: Inglês

10.1074/jbc.m900346200

1083-351X

Bilal Amarneh, Krista A. Matthews, Robert B. Rawson,

Invertebrate Immune Response Mechanisms

During larval development in Drosophila melanogaster, transcriptional activation of target genes by sterol regulatory element-binding protein (dSREBP) is essential for survival. In all cases studied to date, activation of SREBPs requires sequential proteolysis of the membrane-bound precursor by site-1 protease (S1P) and site-2 protease (S2P). Cleavage by S2P, within the first membrane-spanning helix of SREBP, releases the transcription factor. In contrast to flies lacking dSREBP, flies lacking dS2P are viable. The Drosophila effector caspase Drice cleaves dSREBP, and cleavage requires an Asp residue at position 386, in the cytoplasmic juxtamembrane stalk. The initiator caspase Dronc does not cleave dSREBP, but animals lacking dS2P require both drice and dronc to complete development. They do not require Dcp1, although this effector caspase also can cleave dSREBP in vitro. Cleavage of dSREBP by Drice releases the amino-terminal transcription factor domain of dSREBP to travel to the nucleus where it mediates the increased transcription of target genes needed for lipid synthesis and uptake. Drice-dependent activation of dSREBP explains why flies lacking dS2P are viable, and flies lacking dSREBP itself are not. During larval development in Drosophila melanogaster, transcriptional activation of target genes by sterol regulatory element-binding protein (dSREBP) is essential for survival. In all cases studied to date, activation of SREBPs requires sequential proteolysis of the membrane-bound precursor by site-1 protease (S1P) and site-2 protease (S2P). Cleavage by S2P, within the first membrane-spanning helix of SREBP, releases the transcription factor. In contrast to flies lacking dSREBP, flies lacking dS2P are viable. The Drosophila effector caspase Drice cleaves dSREBP, and cleavage requires an Asp residue at position 386, in the cytoplasmic juxtamembrane stalk. The initiator caspase Dronc does not cleave dSREBP, but animals lacking dS2P require both drice and dronc to complete development. They do not require Dcp1, although this effector caspase also can cleave dSREBP in vitro. Cleavage of dSREBP by Drice releases the amino-terminal transcription factor domain of dSREBP to travel to the nucleus where it mediates the increased transcription of target genes needed for lipid synthesis and uptake. Drice-dependent activation of dSREBP explains why flies lacking dS2P are viable, and flies lacking dSREBP itself are not. Genetic systems offer powerful tools for understanding the role of proteolysis in normal physiology. Studies of mutants lacking one or more proteases can reveal physiologically relevant details not accessible from biochemical approaches alone. One proteolytic signaling pathway that has yielded to the combination of genetics and biochemistry is the sterol regulatory element-binding protein (SREBP) 3The abbreviations used are: SREBP, sterol regulatory element-binding protein; dSREBP, Drosophila SREBP; S1P, site-1 protease; S2P, site-2 protease; ER, endoplasmic reticulum; HSV, herpes simplex virus; YFP, yellow fluorescent protein; GFP, green fluorescent protein; RNAi, RNA interference; AEL, after egg laying. pathway that plays a central role in the regulation of lipid metabolism (1Goldstein J.L. Rawson R.B. Brown M.S. Arch. Biochem. Biophys. 2002; 397: 139-148Crossref PubMed Scopus (200) Google Scholar). SREBPs are membrane-bound transcription factors found in all animals, from placozoans and cnidarians (2Srivastava M. Begovic E. Chapman J. Putnam N.H. Hellsten U. Kawashima T. Kuo A. Mitros T. Salamov A. Carpenter M.L. Signorovitch A.Y. Moreno M.A. Kamm K. Grimwood J. Schmutz J. Shapiro H. Grigoriev I.V. Buss L.W. Schierwater B. Dellaporta S.L. Rokhsar D.S. Nature. 2008; 454: 955-960Crossref PubMed Scopus (662) Google Scholar, 3Putnam N.H. Srivastava M. Hellsten U. Dirks B. Chapman J. Salamov A. Terry A. Shapiro H. Lindquist E. Kapitonov V.V. Jurka J. Genikhovich G. Grigoriev I.V. Lucas S.M. Steele R.E. Finnerty J.R. Technau U. Martindale M.Q. Rokhsar D.S. Science. 2007; 317: 86-94Crossref PubMed Scopus (1182) Google Scholar) to mammals (4Wang X. Briggs M.R. Hua X. Yokoyama C. Goldstein J.L. Brown M.S. J. Biol. Chem. 1993; 268: 14497-14504Abstract Full Text PDF PubMed Google Scholar). Two membrane-spanning helices anchor the ∼120-kDa precursor form to the membranes of the endoplasmic reticulum (ER). Its large amino- and carboxyl-terminal domains reside in the cytoplasm, whereas a short loop projects into the ER lumen. In the ER membrane, SREBP forms a complex with Scap, a polytopic membrane protein harboring a sterol sensing domain. When cellular demand for lipid rises, SREBP-Scap complexes exit the ER via COPII-coated vesicles and travel to the Golgi apparatus. Once there, SREBP is cleaved at two sites to release the soluble, active amino-terminal transcription factor domain. The site-1 protease (S1P) cuts within the luminal loop separating the two membrane-spanning helices. The membrane-bound amino-terminal fragment, which harbors the transcription factor domain, is the substrate for the site-2 protease (S2P). S2P cleaves SREBP within the first membrane-spanning helix, between Leu and Cys residues, and the intermediate form of SREBP accumulates only in the absence of cleavage by S2P (5Espenshade P.J. Hughes A.L. Annu. Rev. Genet. 2007; 41: 401-427Crossref PubMed Scopus (439) Google Scholar). Drosophila melanogaster has a single SREBP gene, dSREBP (also called HLH-106 (6Theopold U. Ekengren S. Hultmark D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1195-1199Crossref PubMed Scopus (38) Google Scholar)), as well as orthologues of S1P, S2P, and Scap (7Seegmiller A.C. Dobrosotskaya I. Goldstein J.L. Ho Y.K. Brown M.S. Rawson R.B. Dev. Cell. 2002; 2: 229-238Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In mammalian cells and in flies, transcriptional up-regulation of the genes of lipid metabolism by SREBP is essential for survival. Mammalian cells lacking S1P, S2P, or Scap cannot activate SREBP and do not survive unless their culture medium is supplemented with free cholesterol and unsaturated fatty acids (1Goldstein J.L. Rawson R.B. Brown M.S. Arch. Biochem. Biophys. 2002; 397: 139-148Crossref PubMed Scopus (200) Google Scholar). Similarly, Drosophila lacking dSREBP die at the end of second instar but can be rescued by supplementing their diet with the ultimate end products of dSREBP activation, fatty acids (8Kunte A.S. Matthews K.A. Rawson R.B. Cell Metab. 2006; 3: 439-448Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Unlike vertebrates, insects cannot synthesize cholesterol and therefore always have a requirement for sterols in their diet (9Clark A.J. Bloch K. J. Biol. Chem. 1959; 234: 2578-2588Abstract Full Text PDF PubMed Google Scholar, 10Sang J.H. J. Exp. Biol. 1956; 33: 45-72Google Scholar). We have recently shown that, in striking contrast to mammalian cells, for which loss of S2P is lethal, flies lacking dS2P survive rather well. In dS2P mutants, dSREBP continues to be activated. Thus, they exhibit a less severe deficit in the transcription of genes involved in lipid metabolism than larvae lacking dSREBP (8Kunte A.S. Matthews K.A. Rawson R.B. Cell Metab. 2006; 3: 439-448Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11Matthews K.A. Kunte A.S. Tambe-Ebot E. Rawson R.B. Genetics. 2009; 181: 119-128Crossref PubMed Scopus (14) Google Scholar). This explains why they survive and larvae lacking dSREBP do not. In mammalian cells, SREBP can be cleaved also during apoptosis by caspases-3 and -7 (12Pai J.T. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5437-5442Crossref PubMed Scopus (65) Google Scholar, 13Wang X. Zelenski N.G. Yang J. Sakai J. Brown M.S. Goldstein J.L. EMBO J. 1996; 15: 1012-1020Crossref PubMed Scopus (296) Google Scholar). Caspases nearly always cleave following an Asp residue. Caspase cleavage of mammalian SREBP occurs at a cytoplasmic site that lies in the juxtamembrane stalk between the DNA binding domain and the first membrane-spanning helix (14Wang X. Pai J.T. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The transcription factor domain released by caspase cleavage can activate a reporter gene under control of a synthetic SREBP target promoter (15Higgins M.E. Ioannou Y.A. J. Lipid Res. 2001; 42: 1939-1946Abstract Full Text Full Text PDF PubMed Google Scholar), but the physiological significance of cleavage of SREBPs during apoptosis is unknown. The genome of D. melanogaster encodes seven caspases as follows: damm (48C5), dcp1 (59E3), decay (89B18), dream (or strica, 42A8), dredd (1B12–13), drice (99C1), and dronc (or Nc, 67D2). Dronc, Dredd, and Dream are thought to be initiator (or apical) caspases that activate other caspases by cleaving them, whereas Damm, Dcp1, Decay, and Drice are effector (or executioner) caspases that cleave various target proteins. Here we combine biochemical and genetic techniques to investigate whether caspases are responsible for the activation of dSREBP in dS2P mutants. We find that two Drosophila effector caspases, Drice and Dcp1, can cleave dSREBP in vitro and in cultured Drosophila S2 cells undergoing apoptosis. Flies lacking dS2P and drice (but not dcp1) phenocopy loss of dSREBP itself, dying during the second larval instar. Importantly, this synthetic lethality is rescued by supplementing the culture medium with fatty acids, just as seen for dSREBP mutants. The initiator caspase Dronc, which activates Drice, is not able to cleave dSREBP in vitro but is also required by dS2P mutants. Thus Drice can cleave dSREBP and is necessary for its activation in the absence of dS2P. This activation enables the survival of dS2P mutants. Expression Plasmids-The plasmid Mtal-Grim, and plasmids harboring cDNAs of Dcp1, Drice, Dronc, Damm, Decay, Dream, and Dredd, were gifts from John Abrams (University of Texas Southwestern) and were used as a template to synthesize DNA fragments for RNAi synthesis. A genomic construct of dSREBP tagged at its amino terminus with enhanced YFP (Clontech) and also tagged with the HSV epitope was engineered such that tags are inserted in-frame at the unique AscI restriction site described (8Kunte A.S. Matthews K.A. Rawson R.B. Cell Metab. 2006; 3: 439-448Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). This construct, designated YFP-dSREBP, was originally designed for germ line transformation. It proved to be more efficiently expressed in S2 cells than versions tagged with HSV alone and was therefore chosen for many of the studies presented here. Site-directed mutagenesis using PCR and/or the QuickChange site-directed mutagenesis kit (Stratagene, Cedar Creek, TX) was employed to construct desired mutations, and the mutagenesis was confirmed by multiple sequencing runs for each strand. Germ line transformation of Drosophila was performed by BESTGENE (Chino Hills, CA). Cell Culture-Transfection of S2 cells was performed as described (7Seegmiller A.C. Dobrosotskaya I. Goldstein J.L. Ho Y.K. Brown M.S. Rawson R.B. Dev. Cell. 2002; 2: 229-238Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). For RNAi studies, S2 cells transfected with Mtal-Grim using Cellfectin (Invitrogen) on day 0 were treated with dsRNA on day 1 for 5 h (16Dobrosotskaya I.Y. Seegmiller A.C. Brown M.S. Goldstein J.L. Rawson R.B. Science. 2002; 296: 879-883Crossref PubMed Scopus (265) Google Scholar). Apoptotic Induction-Cells were treated with 0.7 mm CuSO4 in IPL41 (Invitrogen) medium for 5 h prior to harvest. SDS-PAGE, and immunoblot analysis was performed as described (7Seegmiller A.C. Dobrosotskaya I. Goldstein J.L. Ho Y.K. Brown M.S. Rawson R.B. Dev. Cell. 2002; 2: 229-238Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), using anti-HSV antibody (Novagen) at 1:10,000 dilution. Caspase in Vitro Assays-Recombinant Dcp1 and Drice were the generous gift from Dr. Xiaodong Wang (University of Texas Southwestern). Membrane fractions of HSV and enhanced YFP-tagged dSREBPs were purified from transfected S2 cells as described (7Seegmiller A.C. Dobrosotskaya I. Goldstein J.L. Ho Y.K. Brown M.S. Rawson R.B. Dev. Cell. 2002; 2: 229-238Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The assays were performed in caspase reaction buffer (0.15 ml of 25 mm Tris, 25 mm NaCl, 2 mm dithiothreitol, pH 7.4) containing 100 μg (protein) of membrane fractions and 200 nm caspase. The reaction was incubated up to 60 min at 25 °C and was stopped by addition of sample loading buffer and boiling in preparation for SDS-PAGE. Genetic Strains-All marker mutations and balancer chromosomes are described in and referenced by FlyBase (17FlyBase ConsortiumNucleic Acids Res. 2003; 31: 172-175Crossref PubMed Scopus (383) Google Scholar). Crosses were maintained at 25 °C in vials containing freshly yeasted cornmeal/molasses/agar (8Kunte A.S. Matthews K.A. Rawson R.B. Cell Metab. 2006; 3: 439-448Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) except where noted. OreR flies served as wild type. dS2P1 is a deletion encompassing the dS2P locus, and dS2P2 is a transposon insertion within the dS2P open reading frame as described previously (11Matthews K.A. Kunte A.S. Tambe-Ebot E. Rawson R.B. Genetics. 2009; 181: 119-128Crossref PubMed Scopus (14) Google Scholar). dSREBP189 is a deletion extending into the open reading frame of dSREBP isolated in a screen for imprecise excisants of a nearby P element (8Kunte A.S. Matthews K.A. Rawson R.B. Cell Metab. 2006; 3: 439-448Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Caspase mutants dcp1Prev1 (18Laundrie B. Peterson J.S. Baum J.S. Chang J.C. Fileppo D. Thompson S.R. McCall K. Genetics. 2003; 165: 1881-1888PubMed Google Scholar), driceΔ1 (19Muro I. Berry D.L. Huh J.R. Chen C.H. Huang H. Yoo S.J. Guo M. Baehrecke E.H. Hay B.A. Development (Camb.). 2006; 133: 3305-3315Crossref PubMed Scopus (120) Google Scholar), and dronc51 (20Chew S.K. Akdemir F. Chen P. Lu W.J. Mills K. Daish T. Kumar S. Rodriguez A. Abrams J.M. Dev. Cell. 2004; 7: 897-907Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), were from Kimberly McCall, Bruce Hay, and John Abrams, via the Abrams Laboratory. The P{dSREBPg} and P{dSREBPg(D386A)} transgenes used are inserted on the 2nd chromosome. dS2P, Caspase Double Mutant Assays-Drosophila S2P and caspase double mutant lines were maintained as heterozygous stocks as follows: dS2P/CyO, twist-GFP; driceΔ1/TM3, Ser, actin-GFP, and dS2P/CyO, twist-GFP; dronc51/TM3, Ser, actin-GFP. All crosses were set up using virgin females homozygous for dS2P and heterozygous for drice or dronc. These animals survived poorly on unsupplemented medium (see below). Thus, to collect a sufficient numbers of females, embryos from the heterozygous stocks were plated onto dishes containing 14:0 + 18:1 medium (semi-defined medium supplemented with 0.075% myristate and 0.15% oleate (w/v)). Third instar larvae homozygous for dS2P and heterozygous for drice or dronc were scored using the twist- and actin-GFP markers and transferred to vials containing regular/cornmeal/molasses agar medium. Adult females were collected as they emerged. Virgin dS2P2/dS2P2; driceΔ1 (or dronc51)/TM3, Ser, actin-GFP females were crossed to dS2P1/CyO, twist-GFP; driceΔ1 (or dronc51)/TM3, Ser, actin-GFP males. On day 0, embryos from an overnight collection were plated at 10 mg of embryos/dish onto duplicate 60-mm dishes containing semi-defined medium with no additions ("No Addition") and 60-mm dishes containing 14:0 + 18:1 medium. Two days later, larvae from one "No Addition" and one "14:0 + 18:1" plate were separated from the food by floatation on a salt cushion and scored using the twist- and actin-GFP markers. On day 4, this procedure was repeated for the remaining two dishes. To calculate the percent expected, the observed ratio (calculated by dividing the number of larvae for each genotype by the expected total) was divided by the predicted Mendelian ratio. The total number of larvae expected was calculated based on the emergence of the doubly heterozygous mutants, which are expected to comprise 1/3 of the larvae because of embryonic lethality of animals homozygous for the third chromosome balancer. Thus, the expected total is (number of doubly heterozygous larvae/0.33). Rescue of dSREBP189 Lethality by Genomic dSREBP Transgenes-Independent insertions of P{dSREBPg(D386A)} on the second chromosome were recombined onto both dS2P1 and dS2P2 alleles and then crossed into a dSREBP189 background. Recombinants were genotyped by PCR analysis and sequencing of the P{dSREBPg(D386A)} transgene. Lines homozygous or heterozygous for the second chromosome were generated as follows: dS2P, P{dSREBPg(D386A)}/dS2P, P{dSREBPg(D386A)}; dSREBP189/TM6B, Tb Hu e and dS2P, P{dSREBPg(D386A)}/CyO, twist-GFP; dSREBP189/TM6B, Tb Hu e. Virgin dS2P2, P{dSREBPg(D386A)}/dS2P2, P{dSREBPg-(D386A)}; dSREBP189/TM6B, Tb Hu e females were crossed to dS2P1, P{dSREBPg(D386A)}/CyO, twist-GFP; dSREBP189/TM6B, Tb Hu e males. On day 0, embryos from an overnight collection were seeded into 10 vials (at 1 mg embryos/vial) containing regular/cornmeal/molasses agar (No Addition) and 10 vials containing regular/cornmeal/molasses agar supplemented with 0.075% myristate and 0.15% oleate. Emerging adults were scored using twist-GFP and Hu markers until no further adults emerged (approximately day 19 AEL). The data are presented as percent of the expected calculated as described above. Statistical Procedures-Data were analyzed using χ2 tests for independence and goodness of fit. The Sequential Bonferroni Correction of Holms was applied to pairwise comparisons. dSREBP Is Cleaved during Apoptosis in Drosophila S2 Cells-Mammalian SREBPs were among the first experimentally verified substrates for cleavage by caspases (14Wang X. Pai J.T. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Therefore, to identify proteases other than dS1P and dS2P that can cleave dSREBP in dS2P– larvae, we first tested the hypothesis that dSREBP can be cleaved by Drosophila caspases. To increase caspase activity in Drosophila S2 cells, we induced apoptosis by expression of the apoptotic activator Grim under control of the inducible metallothionine promoter (21Chen P. Nordstrom W. Gish B. Abrams J.M. Genes Dev. 1996; 10: 1773-1782Crossref PubMed Scopus (365) Google Scholar). Following addition of copper sulfate (CuSO4), caspase activity in Grim-transfected cells increased dramatically as monitored by cleavage of the fluorescent substrate Ac-DEVD-aminomethylcoumarin (data not shown). Fig. 1A shows a schematic of SREBP topology and the relationship of the various cleavage fragments. We cotransfected S2 cells with Grim and with tagged versions of dSREBP (YFP-dSREBP), either wild type or harboring specific mutations that block cleavage by dS1P and dS2P (7Seegmiller A.C. Dobrosotskaya I. Goldstein J.L. Ho Y.K. Brown M.S. Rawson R.B. Dev. Cell. 2002; 2: 229-238Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The YFP-dSREBP constructs show similar levels of the precursor form ("P") in the membrane fraction (Fig. 1B, upper panel, lanes 1–8). For dSREBP with wild type sequences at site-1 and site-2 (YFP-dSREBP WT), the nuclear form of dSREBP ("N") is detected in both the absence and presence of CuSO4 (Fig. 1B, lower panel, lanes 1 and 2). A novel, more rapidly migrating band (hereafter designated "fragment C") also appears in the nuclear extract of the CuSO4-treated sample (lane 2) but not the untreated one (lane 1). Mutating the crucial Arg at position 486 in site-1 to Ala blocks cleavage by dS1P (7Seegmiller A.C. Dobrosotskaya I. Goldstein J.L. Ho Y.K. Brown M.S. Rawson R.B. Dev. Cell. 2002; 2: 229-238Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), and no nuclear dSREBP is observed in the absence of CuSO4 (Fig. 1B, lower panel, lane 3). The smaller, novel fragment C appears when apoptosis is induced (Fig. 1B, lower panel, lane 4). Mutating an Asp-Pro motif within the first membrane-spanning helix of dSREBP blocks cleavage by dS2P but leaves cleavage at site-1 unaffected (7Seegmiller A.C. Dobrosotskaya I. Goldstein J.L. Ho Y.K. Brown M.S. Rawson R.B. Dev. Cell. 2002; 2: 229-238Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The intermediate form ("I") of dSREBP is the product of dS1P cleavage and remains attached to the membrane because it retains a single membrane-spanning helix. The intermediate form appears in both the presence and absence of CuSO4 (Fig. 1B, upper panel, lanes 5 and 6). The normal nuclear form is absent from the nuclear extract (Fig. 1B, lower panel, lanes 5 and 6), but fragment C appears when apoptosis is induced (Fig. 1B, lower panel, lane 6). The same pattern holds true when both site-1 and -2 are mutated (Fig. 1B, lower panel, lanes 7 and 8). These results parallel results using versions of dSREBP tagged with the HSV epitope alone (data not shown). Thus, dSREBP can be cleaved when caspase activity is up-regulated in S2 cells, and this cleavage does not require prior cleavage by dS1P or dS2P. Consistent with this finding, caspase cleavage of dSREBP during apoptosis is unaffected by treating S2 cells with palmitate and ethanolamine (data not shown), which suppresses cleavage of dSREBP by dS1P and dS2P (16Dobrosotskaya I.Y. Seegmiller A.C. Brown M.S. Goldstein J.L. Rawson R.B. Science. 2002; 296: 879-883Crossref PubMed Scopus (265) Google Scholar). dSREBP Is Cleaved by Drice and Dcp1-To investigate further the role of caspases in the production of dSREBP fragment C, we performed RNAi experiments in Drosophila S2 cells targeting dronc, dredd, decay, drice, dream, damm, and dcp1 and determined whether fragment C still was produced upon induction of apoptosis (supplemental Fig. 1). When apoptosis was induced by expression of Grim, RNAi against Drice blocked the appearance of fragment C (supplemental Fig. 1, lane 7), whereas RNAi against Dcp1 partially reduced the accumulation of fragment C (supplemental Fig. 1, lane 10). We consistently observed the partial effect of RNAi against Dcp1 and the full effect of Drice RNAi. The evident effect of RNAi treatment against Drice and Dcp1 indicates that at least these two effector caspases can mediate cleavage of dSREBP during Grim-induced apoptosis in S2 cells. To test whether Drice and Dcp1 can cleave dSREBP directly, we prepared membrane fractions from S2 cells transfected with YFP-dSREBP and incubated these membranes in vitro with purified recombinant Drice or Dcp1 (Fig. 2). At 0 min, no caspase cleavage product is observed with either protease (Fig. 2, lanes 1 and 5). Increasing accumulation of fragment C is observed over time (Fig. 2, lanes 2–4 and 6–8). No product is observed at 60 min in the absence of added protease (Fig. 2, lane 10). These data demonstrate that Drice and Dcp1 can cleave membrane-bound dSREBP in vitro. Dronc Does Not Cleave dSREBP-We also tested the ability of the initiator caspase Dronc to cleave dSREBP, using purified recombinant Dronc. These results are shown in Fig. 3. Incubation of membranes containing wild type dSREBP with either Drice or Dcp1 resulted in substantial accumulation of fragment C (Fig. 3, lanes 11 and 12). No accumulation of fragment C was observed with purified Dronc (Fig. 3, lane 4). Dronc can cleave Ac-DEVD-amidotrifluoromethylcoumarin (22Hawkins C.J. Yoo S.J. Peterson E.P. Wang S.L. Vernooy S.Y. Hay B.A. J. Biol. Chem. 2000; 275: 27084-27093Abstract Full Text Full Text PDF PubMed Google Scholar, 23Dorstyn L. Kumar S. Cell Death Differ. 2008; 15: 461-470Crossref PubMed Scopus (49) Google Scholar). When we introduced that cleavage site motif into the juxtamembrane region of dSREBP (383FTTDAGL was mutated to 383FTTDEVD), incubation with Dronc resulted in accumulation of fragment C (Fig. 3, lane 8), confirming that the enzyme had activity in this assay. With the mutant Dronc-cleavable substrate, accumulation of fragment C was blocked by the addition of the caspase inhibitor Ac-DEVD-CHO (Fig. 3, lane 9). Recombinant Dronc can cleave a version of dSREBP containing a motif that Dronc has been shown to cleave but does not cleave wild type dSREBP. Thus, the effector caspases Drice and Dcp1 cleave dSREBP, but the initiator caspase Dronc does not. Cleavage of dSREBP by Caspases Requires an Asp Residue at Position 386-As their name indicates (24Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2158) Google Scholar), caspases have a decided preference for an Asp residue in the P1 position. Drice and Dcp1 have cleavage specificities similar to mammalian caspase 3, with the sequence DEVD reported to be optimal for cleavage by Drice in vitro (25Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (127) Google Scholar). Fig. 4A shows sequence alignment of the juxtamembrane stalk region (between the transcription factor domain and the first membrane-spanning helix) of SREBPs from each of the 12 SREBP sequences available from the genus Drosophila. There are four Asp residues within this region of dSREBP (Fig. 4A, black boxes), and these four are conserved among all species, save Drosophila willistoni. We note that many other attributes of the genome sequence of this species are also exceptional among the 12 species sequenced (26Clark A.G. Eisen M.B. Smith D.R. Bergman C.M. Oliver B. Markow T.A. Kaufman T.C. Kellis M. Gelbart W. Iyer V.N. Pollard D.A. Sackton T.B. Larracuente A.M. Singh N.D. Abad J.P. et al.Nature. 2007; 450: 203-218Crossref PubMed Scopus (1555) Google Scholar, 27Vicario S. Moriyama E.N. Powell J.R. BMC Evol. Biol. 2007; 7: 226Crossref PubMed Scopus (160) Google Scholar). A multiple phylum alignment of this region is shown in supplemental Fig. 2. We individually mutated each of these Asp residues to Ala in the context of YFP-dSREBP, and we assessed the ability of each construct to be cleaved during apoptosis (Fig. 4B). The apoptosis-dependent fragment C appears with wild type dSREBP (Fig. 4B, lanes 2 and 12) and with Asp to Ala mutants at positions 395, 398, and 407 (lanes 6, 8 and 10). No fragment is observed when the Asp at 386 is mutated to Ala (Fig. 4B, lane 4). The dependence of cleavage on an Asp at 386 (but not on any other Asp residue in this region) indicates that cleavage during apoptosis occurs at the sequence 383FTTD↓A387 in dSREBP, where the down arrow indicates the scissile bond. Consistent with this conclusion, in transfected S2 cells, fragment C comigrates with a truncated version of dSREBP that has a stop codon in place of residue 387 (data not shown). This was somewhat surprising as Drice and Dcp1 are thought to prefer the sequence DXXD as is present at 395. We therefore further tested the requirement of Asp-386 for in vitro cleavage by Drice by incubating membrane fractions from S2 cells transfected with YFP-dSREBP, either wild type or harboring the D386A mutation (Fig. 5A). Accumulation of fragment C proceeded in a time-dependent fashion with the wild type substrate (Fig. 5A, lanes 3–6), but no fragment C was produced from the D386A mutant (lanes 7–10). In S2 cells, cleavage during apoptosis was also abolished when Asp-386 was substituted by Glu or Asn (Fig. 5B, lanes 6 and 8). The supplemental Fig. 3 shows that the threonine residue at position 385 is also required for cleavage (lanes 4 and 6). Even when a serine is introduced at this position, cleavage is abolished (supplemental Fig. 3, lane 4). This Thr residue is as well conserved among Drosophila as Asp-386, whereas residues in the P3 and P4 positions are not highly conserved (Fig. 4A). Consistent with this evolutionarily acceptable variation, we find that a T384A mutant is cleaved as readily as wild type (data not shown). Drice Is Required for the Survival of dS2P Mutant Larvae-In S2 cells and using purified enzyme in vitro, Drice and Dcp1 can cleave dSREBP, and cleavage is blocked by substituting Asp-386 with Ala. We hypothesized that this cleavage of dSREBP by caspases is responsible for the survival of flies lacking dS2P. To test this, we constructed stocks harboring null mutations both in dS2P as well as in one each of the two caspases that were shown to cleave dSREBP, using the alleles dcp1Prev1 and driceΔ1 (18Laundrie B. Peterson J.S. Baum J.S. Chang J.C. Fileppo D. Thompson S.R. McCall K. Genetics. 2003; 165: 1881-1888PubMed Google Scholar, 19Muro I. Berry D.L. Huh J.R. Chen C.H. Huang H. Yoo S.J. Guo M. Baehrecke E.H. Hay B.A. Development (Camb.). 2006; 133: 3305-3315Crossref PubMed Scopus (120) Google Scholar). If cleavage of dSREBP by one of these caspases were responsible for the survival of dS2P mutants, then flies lacking both that caspase and dS2P would be unable to activate dSREBP. The doubly mutant animals should evince the phenotype observed in flies completely lacking dSREBP (dSREBP189) and die at the end of second larval instar because of deficient transcription of dSREBP target genes. Importantly, if this "synthetic" lethality were indeed because of deficient dSREBP activation, then supplementing the larval diet with free fatty acids should afford substantial rescue of the dS2P, drice double mutants, just as seen for dSREBP mutants (8Kunte A.S. Matthews K.A. Rawson R.B. Cell Metab. 2006; 3: 439-448Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Flies homozygous for dcp1Prev1 are viable (18Laundrie B. Peterson J.S. Baum J.S. Chang J.C. Fileppo D. Thompson S.R. McCall K. Genetics. 2003; 165: 1881-1888PubMed