CED-4 induces chromatin condensation in Schizosaccharomyces pombe and is inhibited by direct physical association with CED-9.
BACKGROUND
Three principal genes are involved in developmental programmed cell death (PCD) in the nematode worm Caenorhabditis elegans. The ced-3 and ced-4 genes are both required for each PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes in cells that are destined to survive. Vertebrate homologues of both ced-3 and ced-9 have been identified as the genes encoding the caspase cysteine proteases and the Bcl-2 family, respectively. In contrast, no vertebrate homologue of ced-4 is known. The CED-3/caspases are important effectors of apoptosis that are presumed to act by cleaving specific target substrates. However, the molecular functions of the CED-9/Bcl-2 and CED-4 proteins are unknown. The unicellular yeast Schizosaccharomyces pombe shares many general cellular properties with metazoa, but has no identified cell suicide machinery. We have therefore used S. pombe as a naive model cell system in which to examine the biological effects of cell-death proteins.


RESULTS
Induction of wild-type ced-4 expression in S. pombe resulted in rapid focal chromatin condensation and lethality. Mutation of the putative nucleotide-binding P-loop motif of CED-4 (K165Q) eliminated the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggested that the phenotype may result from an intrinsic activity of CED-4. Co-expression of ced-9 prevented CED-4-induced chromatin condensation and lethality, and caused the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis.


CONCLUSIONS
Using S. pombe as a model system in which to assay CED-4 function, we have identified a potential direct role for CED-4 in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. The CED-9-mediated rescue of CED-4-induced lethality in this system and the interaction of the two proteins in the yeast two-hybrid analysis suggest that CED-9 inhibits CED-4 action by direct physical association.
James
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Imperial Cancer Research Fund Laboratories, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
Gschmeissner
S
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Fraser
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A
Evan
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GI
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins / Q000096:biosynthesis*; D002843:Chromatin / Q000502:physiology* / Q000648:ultrastructure*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012568:Schizosaccharomyces / Q000502:physiology* / Q000648:ultrastructure; D014714:Vertebrates
D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; C089224:Ced-4 protein, C elegans; C087233:Ced-9 protein, C elegans; D002843:Chromatin; D015801:Helminth Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins