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First published online 30 May 2006
doi: 10.1242/jcs.02985

Journal of Cell Science 119, 2572-2582 (2006)
Published by The Company of Biologists 2006
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Human Bcl-2 cannot directly inhibit the Caenorhabditis elegans Apaf-1 homologue CED-4, but can interact with EGL-1

A. M. Jabbour1,2,3, M. A. Puryer1,2, J. Y. Yu4, T. Lithgow5, C. D. Riffkin1,2, D. M. Ashley1,2,3, D. L. Vaux4,6, P. G. Ekert1,2,7 and C. J. Hawkins1,2,3,6,*

1 Children's Cancer Centre, Royal Children's Hospital, Parkville 3052, Australia
2 Murdoch Children's Research Institute, Royal Children's Hospital, Parkville 3052, Australia
3 Department of Paediatrics, University of Melbourne, Parkville 3010, Australia
4 Walter and Eliza Hall Institute, Royal Melbourne Hospital, Parkville 3050, Australia
5 Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville 3010, Australia
6 Department of Biochemistry, La Trobe University, Bundoora 3086, Australia
7 Department of Neonatology, Royal Children's Hospital, Parkville 3052, Australia


Figure 1
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  		Fig. 1. Reconstitution of the core nematode PCD pathway in S. cerevisiae. (A) A semi-quantitative assay compares the effect of transgenes on yeast viability and growth. Yeast were transformed with the indicated plasmids, directing expression of either wild-type or mutant components of the core apoptosis pathway under the control of either a galactose-inducible promoter (GALS) or constitutive promoter (ADH). Suspensions of each transformant were prepared at standardised concentrations. Serial dilutions were made and spotted onto solid inducing minimal media (galactose) vertically down the plate. Colony size indicates growth rate and colony number reflects cell viability. Each dilution was also spotted onto a repressing plate (glucose) to verify that equivalent numbers of each transformant were spotted; only dilutions four and five are shown. (B) Fusion of a GFP tag to CED-4, a FLAG tag to CED-9, and a myc tag to EGL-1 did not affect function. The specified plasmids were transformed into yeast and assayed as described above. Immunoblotting was performed to visualise expression of epitope-tagged proteins. An asterisk indicates a non-specific yeast protein recognised by the anti-GFP antibody. Coomassie staining allowed visualisation of protein loading.

 

Figure 2
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  		Fig. 2. Unlike CED-9, Bcl-2 cannot inhibit CED-4-dependent yeast death. (A) Yeast were transformed and spotted as described in the legend to Fig. 1. The ability of Bcl-2 to inhibit Bax-dependent and CED-4-dependent yeast death was tested, using Bcl-2 expressed from either the same promoter as CED-9 (ADH) or a very strong galactose-inducible promoter (GALL). The resulting expression levels were visualised by anti-Bcl-2 immunoblotting. Immunoblotting was also performed to monitor expression of GFP-tagged CED-4 and FLAG-tagged CED-9. An asterisk indicates a crossreacting yeast protein recognised by the anti-GFP antibody. Coomassie staining indicates protein loading. (B) Lowering expression of CED-4 using a methionine-repressible promoter (MET) yielded only weak CED-4-dependent yeast death upon co-expression with CED-3. Bcl-2 expression, directed by the intermediate-strength (ADH) or very strong (GALL) promoters, could not suppress even this weak death stimulus. (C) The two-hybrid yeast strain HF7c was transformed with the indicated plasmids or empty vector controls. Transformants were spotted onto minimal medium either containing histidine (growth confirms the presence of the plasmids) or lacking histidine (growth indicates interaction between the prey and bait proteins).

 

Figure 3
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  		Fig. 3. Unlike CED-9, Bcl-2 does not alter CED-4 localisation in yeast. Yeast were transformed with the indicated plasmids. Transformants were either stained for nucleic acid using DAPI (blue; upper three rows) or mitochondria using MitoTracker (red; middle three rows). The endoplasmic reticulum was visualised by anti-Kar2p immunofluorescence (red; lower three rows). GFP-tagged CED-4, CED-9 and Bcl-2 were detected (green fluorescence). GFP-CED-4 localisation was also observed in the presence of either FLAG-CED-9 or Bcl-2. Bars, 5 µm.

 

Figure 4
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  		Fig. 4. Bcl-2 does not cooperate with CED-9 to inhibit CED-4-dependent yeast death. Yeast were transformed with the specified plasmids and spotted on inducing or repressing medium, as described in the legend to Fig. 1. FLAG-tagged CED-9 was expressed either by the constitutive ADH promoter or an inducible copper promoter (CUP1). The copper concentration in the inducing medium was varied to control FLAG-CED-9 expression levels in yeast transformed with the pCUP1-(TRP1)-FLAG-CED-9 plasmid. In the absence of added copper, only weak protection was afforded. The impact of Bcl-2 on this weak protection was tested by co-expressing Bcl-2, either using the intermediate-strength promoter (ADH) or the very strong promoter (GALL). Transformants were also grown in liquid galactose-containing media with no added copper. Lysates generated from those cultures were immunoblotted to visualise FLAG-CED-9 and Bcl-2 levels. Coomassie staining allowed visualisation of protein loading.

 

Figure 5
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  		Fig. 5. Bcl-2 interacts with EGL-1. (A) The two-hybrid yeast strain HF7c was transformed with the indicated plasmids or empty vector controls. Transformants were spotted (as described in the legend to Fig. 1) onto minimal medium either containing histidine (growth confirms the presence of the plasmids) or lacking histidine (growth indicates interaction between the prey and bait proteins). (B) In vitro translated [35S]methionine-labelled FLAG-Tab-1, [35S]methionine-labelled CED-9 or [35S]methionine-labelled Bcl-2 were incubated with bacterial lysates containing recombinant HIS-tagged p35, HIS-tagged EGL-1 or HIS-tagged CED-4. The protein complexes were then bound to Ni-NTA beads and subjected to SDS-PAGE. [35S]-labelled proteins, bound through the recombinant proteins to the beads, were detected (upper panel). The complexes were also immunoblotted to detect the HIS-tagged proteins (lower panel). The arrow denotes full-length CED-4-6HIS. The asterisk indicates a smaller protein that might be a breakdown product. (C) Yeast were transformed with the indicated plasmids and spotted onto either repressing or inducing media as described in Fig. 1. Immunoblotting was used to detect Bax, Bcl-2, Bim and myc-EGL-1. Coomassie staining illustrates protein loading.

 

Figure 6
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  		Fig. 6. CED-9 and Bcl-2 relocalise EGL-1 in yeast. Yeast expressing GFP-EGL-1 were transformed with the indicated plasmids. Transformants were either stained for nucleic acid with DAPI (blue; upper three rows) or mitochondria using MitoTracker (red; middle three rows). The endoplasmic reticulum was visualised by anti-Kar2p immunofluorescence (red; lower three rows). GFP-tagged EGL-1 localisation was monitored (green fluorescence) in the presence and absence of either FLAG-CED-9 or Bcl-2. Bars, 5 µm.

 

Figure 7
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  		Fig. 7. Models for the mechanism by which transgenic Bcl-2 might inhibit nematode PCD. (A) The core nematode PCD pathway is depicted. (B) The previously presumed model for the inhibition of nematode cell death by Bcl-2. Data presented here indicate that Bcl-2 cannot bind CED-4 nor inhibit its activity, arguing against this model. (C) Our results suggest this model, in which Bcl-2 overexpression might provide an alternative `sink' for EGL-1, thereby preventing EGL-1 from accessing CED-9. CED-9 would then be available to inhibit CED-4 and thus suppress PCD.

 

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© The Company of Biologists Ltd 2006