Activation of sterol regulatory element-binding protein by the caspase Drice in Drosophila larvae

Abstract

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.

FIGURE 1.

Cleavage of dSREBP during apoptosis in S2 cells. A, this schematic shows SREBP topology. bHLH designates the transcription factor domain. The gray bar represents the lipid bilayer. Arrows indicate the site-1 protease and site-2 protease cleavage sites. The cytoplasmic juxtamembrane stalk region is indicated by the brace. NH₂ and COO^– designate the amino and carboxyl termini. The various cleavage products are illustrated below. I, intermediate form; N, normal nuclear form; C, caspase-dependent band. B, cells were cotransfected with the indicated YFP-dSREBP constructs and with Mtal-Grim as described under “Experimental Procedures.” We added 0.7 mm CuSO₄ to induce expression of Grim. Cells were fractionated, and the membrane pellet was washed with 0.1 m sodium carbonate prior to immunoblot analysis using an anti-HSV antibody as described under “Experimental Procedures.” P, precursor; I, intermediate form; N, normal nuclear form; C, caspase-dependent band; WT, wild type.

FIGURE 2.

Cleavage of dSREBP by Drice and Dcp1 in vitro. Membrane fractions were purified from S2 cells transfected with YFP-dSREBP as described. The membranes were incubated along with the indicated purified recombinant caspase in caspase reaction buffer. The reaction was stopped at the indicated times as described. The membranes were then subject to immunoblot analysis using an anti-HSV antibody. P, precursor; C, caspase-dependent band; I, intermediate form.

FIGURE 3.

The effector caspase Dronc does not cleave dSREBP. Membrane fractions were prepared from S2 cells transfected with the indicated plasmids and incubated with the indicated enzyme in caspase buffer for the times indicated. Samples shown in lanes 5 and 9 were treated with the caspase inhibitor Ac-DEVD-CHO (25 μm, Cayman Chemicals, Ann Arbor, MI) at the time of enzyme addition. Whole cell lysates from apoptotic S2 cells transfected with YFP-dSREBP and MTAL-grim (lanes 1 and 2) are shown for comparison of cleaved fragments. Samples were subjected to immunoblot analysis using anti-HSV antibody as described under “Experimental Procedures.” P, precursor; C, caspase-dependent band; I, intermediate form; WT, wild type.

FIGURE 4.

Caspase cleavage of dSREBP in cultured cells requires Asp-386. A, sequence alignment of SREBP homologues from 12 Drosophila species. In the abbreviated names, “mel” indicates melanogaster (CG8522-PA); sim, simulans (GD14825-PA); sec, sechellia (GM19644-PA), yak, yakuba (GE19622-PA), ere, erecta (GG16056-PA); ana, anassae (GF23590-PA); per, persimilis (GL15732-PA); pse, pseudoobscura (GA21134-PA); gri, grimshawii (GH14653-PA), wil, willistoni (GK17496-PA); vir, virilis (GJ11320-PA); and moj, mojavensis (GI11638-PA). Sequences were aligned using the ClustalW algorithm. Asp residues are shaded black. B, Drosophila S2 cells were transfected with constructs encoding wild type YFP-dSREBP or YFP-dSREBP harboring an Ala in place of Asp residues at, respectively, positions 386, 395, 398, or 407 in the juxtamembrane stalk. Expression of Grim was induced with CuSO₄, and whole cell lysates were subjected to immunoblot analysis using anti-HSV antibody. P, precursor; N, normal nuclear form; C, caspase-dependent band.

FIGURE 5.

Cleavage by Drice in vitro requires an Asp at position 386. A, in vitro cleavage of dSREBP requires Asp-386. Membrane fractions were purified from S2 cells transfected with YFP-dSREBP or YFP-dSREBP(D386A). Membranes were incubated for the indicated times in caspase reaction buffer in the presence or absence of purified, recombinant Drice. The reaction was stopped at the indicated times. The membranes were then subject to immunoblot analysis using an anti-HSV antibody. B, in vivo cleavage of dSREBP requires Asp-386. S2 cells were cotransfected with Mtal-grim and the indicated YFP-dSREBP construct, and the experiment was conducted as described in the legend to Fig. 1. Note that two intervening lanes between lanes 8 and 9 have been omitted for clarity. P, precursor; N, normal nuclear form; C, caspase-dependent band; I, intermediate form.

FIGURE 6.

Drice and Dronc are essential for the survival of larvae lacking dS2P. A, virgin females homozygous for dS2P² were crossed to males heterozygous for dS2P¹. Parallel cultures were inoculated with 10 mg/plate of embryos on regular or supplemented medium (14:0 + 18:1). Larvae were scored on day 2 and day 4 AEL. A cross of dSREBP¹⁸⁹ heterozygotes served as a control for the efficacy of rescue. B, fly lines harboring mutations in both dS2P¹ or dS2P² and in drice^Δ1 were constructed as described under “Experimental Procedures.” Virgin females homozygous for dS2P² and heterozygous for drice^Δ1 were crossed to males heterozygous for dS2P¹ and drice^Δ1. The dS2P homozygous, drice^Δ1 heterozygous females were raised on medium supplemented with fatty acids to enable efficient recovery of these flies. Larvae homozygous or heterozygous for drice^Δ1 and wild type for dS2P survived equally well under all conditions tested in this experiment. C, crosses of flies doubly mutant for dS2P and dronc⁵¹ were conducted as described in B. Horizontal lines corresponding to the value for dSREBP¹⁸⁹ larvae under each condition are shown to facilitate comparison (black, supplemented medium; gray, unsupplemented medium). “+” indicates wild type, and “–” indicates null alleles as described above. Note that the values displayed are for whole populations rather than samples. * indicates p < 0.005 for day 2 compared with day 4. † indicates p < 0.005 for supplemented compared with unsupplemented medium. ‡ indicates p < 0.005 for dS2P transheterozygotes compared with dS2P; caspase larvae. A mean of 1057 larvae were scored for each cross and condition (range = 1446 to 751). The results shown are from a single experiment and are representative of three independent replications.

FIGURE 7.

Survival of dS2P^– mutants requires caspase-cleavable dSREBP. The loci considered are shown at left. For dS2P and dSREBP genotypes, + indicates wild type, and – indicates the null alleles. D₃₈₆A designates the P{dSREBPg(D386A)} transgene. + indicates its presence, and – indicates its absence. dSREBP¹⁸⁹ homozygotes served as a control for the efficacy of dietary rescue in this experiment (far right). Error bars represent the S.E.