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. 1999 Mar 16;96(6):2885-90.
doi: 10.1073/pnas.96.6.2885.

A cloning method to identify caspases and their regulators in yeast: identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1

C J Hawkins  1 , S L Wang, B A Hay
Affiliations

A cloning method to identify caspases and their regulators in yeast: identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1

C J Hawkins et al. Proc Natl Acad Sci U S A. .

Abstract

Site-specific proteases play critical roles in regulating many cellular processes. To identify novel site-specific proteases, their regulators, and substrates, we have designed a general reporter system in Saccharomyces cerevisiae in which a transcription factor is linked to the intracellular domain of a transmembrane protein by protease cleavage sites. Here, we explore the efficacy of this approach by using caspases, a family of aspartate-specific cysteine proteases, as a model. Introduction of an active caspase into cells that express a caspase-cleavable reporter results in the release of the transcription factor from the membrane and subsequent activation of a nuclear reporter. We show that known caspases activate the reporter, that an activator of caspase activity stimulates reporter activation in the presence of an otherwise inactive caspase, and that caspase inhibitors suppress caspase-dependent reporter activity. We also find that, although low or moderate levels of active caspase expression do not compromise yeast cell growth, higher level expression leads to lethality. We have exploited this observation to isolate clones from a Drosophila embryo cDNA library that block DCP-1 caspase-dependent yeast cell death. Among these clones, we identified the known cell death inhibitor DIAP1. We showed, by using bacterially synthesized proteins, that glutathione S-transferase-DIAP1 directly inhibits DCP-1 caspase activity but that it had minimal effect on the activity of a predomainless version of a second Drosophila caspase, drICE.

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Figures

Figure 1
Figure 1
A genetic system for monitoring caspase activity in yeast using a transcriptional reporter. Yeast were created that express a chimeric type-1 transmembrane protein (CLBDG6) in which the N-terminal signal sequence and transmembrane domain (CD4) is followed by a linker consisting of six tetrapeptide caspase target sites (indicated in bold) that bracket the specificity of known caspases and granzyme B (23)—DEVDG-WEHDG-IEHDG-IETDG-DEHDG-DQMDG—each of which is followed by a glycine residue, which acts as a stabilizing residue in the N-end rule degradation pathway in yeast (reviewed in ref. 24). C-terminal to the caspase target site linker is a transcription factor domain, LexA-B42. The LexA-dependent transcriptional reporter consists of LexA binding sites (LexA UAS) and a promoter (P) upstream of the bacterial lacZ gene (lacZ) (A). The cells in A act as caspase activity reporters because expression of an active caspase results in CLBDG6 cleavage at the caspase target sites, releasing LexA-B42, which enters the nucleus and activates lacZ transcription (B). A version of CLBDG6 in which the P1 aspartates are changed to glycines (CLBGG6) cannot be cleaved by caspases. Cells expressing CLBGG6 act as false positive reporters for molecules that activate lacZ expression independent of cleavage at caspase target site (C). If cells in B express a caspase inhibitor as well as an active caspase, caspase activity, and thus caspase-dependent release of LexA-B42, is inhibited, and β-gal levels are decreased compared with cells that express the caspase alone (D).
Figure 2
Figure 2
Yeast expressing CLBDG6 act as reporters for CED-3 caspase activity. W303α yeast were transformed with pSH18–34, which carries a LexA-responsive lacZ transcriptional cassette (the LexA/β-gal reporter strain). These cells were transformed with pGALL expression plasmids carrying CLBDG6 (DG6) or CLBGG6 (GG6). These cells also carry a copper-inducible pCUP1 plasmid, which contains either wild-type CED-3 (CED-3), an inactive C to S mutant version of CED-3 (CED-3 C-S), or nothing. Duplicate colonies from each transformation were streaked onto gal/raf medium to induce GAL1-dependent expression of the caspase substrates and then were lifted onto complete media plates with 3 μM copper sulfate to induce caspase expression. After a 12-hr induction, an X-gal assay was performed on the filter. Only cells expressing CLBDG6 and wild-type CED-3 have significant β-gal activity (A). Cultures from three transformants carrying pSH18–34, pGAL-CLBDG6, and either the empty pCUP1 vector or pCUP1-CED-3 were grown to stationary phase, then were diluted into medium containing the indicated levels of copper sulfate and grown for a further 10 hr. o-nitrophenyl-β-d-galactoside assays were performed, and β-gal activity was determined. β-gal activity in the CED-3-expressing cells increased as a function of copper concentration (filled circles, CED-3). No β-gal activity was found in the cultures carrying only the empty pCUP1 vector (open boxes, vector) (B).
Figure 3
Figure 3
Expression of Apaf-1530 induces caspase 9-dependent reporter activation. W303α containing pSH18–34 and pGALL-CLBDG6 were transformed with pGALL plasmids to carry either two empty pGALL vectors, Apaf-1530 and an empty pGALL vector, caspase 9 and an empty pGALL vector, or Apaf-1530 and caspase 9. Two colonies from each transformation were streaked onto selective glucose medium plates, were grown for several days, and then were replica plated onto complete gal/raf medium. After 16 hr, an X-gal assay was performed. Only cells expressing both Apaf-1530 and caspase 9 show significant β-gal activity.
Figure 4
Figure 4
Expression of caspase inhibitors suppresses caspase-dependent reporter activation in yeast. The LexA/β-gal reporter strain carrying pGALL-CLBDG6 was transformed with either an empty pCUP1 plasmid or pCUP1-CED-3 and either an empty pGALL vector or pGALL-p35. Three colonies from each transformation were grown for 24 hr in selective gal/raf medium. Cultures were diluted 1:10 into fresh gal/raf medium containing 3 μM copper sulfate and were grown for a further 10 hr, after which o-nitrophenyl-β-d-galactoside assays for β-gal activity were performed. Cultures from caspase transformants showed significant β-gal activity, which was suppressed by GALL-dependent expression of baculovirus p35 (A). In an experiment similar to that described in A, expression of caspase 753 was induced in cells that express baculovirus p35 or the mouse IAP MIHA. Expression of caspase 753 resulted in a significant increase in cellular β-gal activity, which was suppressed by GALL-dependent expression of p35 or MIHA (B).
Figure 5
Figure 5
High level expression of the Drosophila caspase DCP-1 kills yeast and is prevented by coexpression of baculovirus p35 or DIAP1. EGY48 yeast were transformed with pGALL plasmids containing either full length DCP-1 (FLDCP-1), an active site C-S mutant of DCP-1 (DCP-1 C-S), or no insert (vector). Transformants were streaked from selective glucose-containing medium onto selective gal/raf-inducing medium. Cells expressing either an empty pGALS vector or the DCP-1 C-S active site mutant grow on galactose-containing medium whereas cells expressing full length DCP-1 do not (A). EGY48 yeast carrying pGALS-FLDCP-1 were transformed with pGALL vectors carrying full length DIAP1 (DIAP1), the DIAP1 BIR repeats (DIAP1 BIR), baculovirus p35 (p35), or nothing (vector). GALL-dependent expression of p35, full length DIAP1, and, to a somewhat lesser extent, the DIAP1 BIR repeats block DCP-1-dependent cell death (B).
Figure 6
Figure 6
GST-DIAP1 inhibits the caspase activity of bacterially synthesized DCP-131-His6, but not drICE81-HIS6. Purified GST-DIAP1 (0.16 μM) (open triangles) or GST (0.48 μM) (open squares) was incubated with a fixed amount of DCP-131His6 (0.2nM) in caspase activity assay buffer containing 100 μM of the Ac-DEVD-AFC substrate. Release of AFC was monitored fluorometrically over time. GST-DIAP1 inhibits DCP-1-dependent caspase activity (A). In similar experiments in which 0.62nM drICE81His6 was incubated in caspase activity buffer with GST (0.48 μM) or GST-DIAP1, (0.16 μM), no inhibition of drICE activity by GST-DIAP1was seen (B).
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