Many genes that play important roles in C. elegans programmed cell death have been identified through genetic approaches. These genes include
ced-9, which prevents the death of cells that normally live, and
ced-3 and
ced-4, which are required for cell death. Genetic studies have placed
ced-4 between
ced-9 and
ced-3 in the cell-death pathway. The ced- 3,
ced-4, and
ced-9 genes have all been cloned. Unlike
ced-3 and
ced-9, which have mammalian homologues,
ced-4 encodes a novel protein with no obvious homology with any known protein, making it difficult to investigate its molecular function. Interestingly, two alternatively- spliced
ced-4 transcripts exist, which encode protein products that differ by the presence or absence of 24 amino acids (S. Shaham and H.R. Horvitz). The ectopic expression of the short transcript, which is more abundant, kills cells, while that of the long transcript protects cells from death, indicating that
ced-4 might have dual roles in cell death. To understand the molecular functions of
ced-4 and to discover new members of the cell-death pathway that are functionally related to ced- 4, we are trying to identify proteins that physically interact with the CED-4 protein using the yeast two-hybrid system, which allows the detection of protein-protein interaction through the activation of reporter gene transcription. From several existing two-hybrid systems, we chose to use the Gal4 DNA binding domain/transcription activation domain system, which has been applied successfully in many screens (1). We plan to screen for C. elegans and mammalian proteins that interact with CED-4. By using proteins encoded by each of the two alternative
ced-4 transcripts as baits, we hope to identify proteins that specifically bind to one but not the other form of CED-4, which will reveal pathways specific to the killing or protective functions of ced- 4. We will study the CED-4-interacting proteins using molecular and genetic tools, focusing on the following aspects: 1) How do CED-4 interacting proteins regulate or transmit
ced-4 function? 2) Do these proteins interact with other members of the C. elegans cell-death pathway? 3) What are the in vivo functions of the genes encoding CED-4 interacting proteins? Are there any existing mutations of these genes? 4) What are the positions of these genes in the genetic pathway for programmed cell death? We hope that the isolation of CED-4 interacting proteins will not only help clarify the multiple functions of
ced-4 but also identify new members of the cell death pathway, thus leading to the further understanding of the molecular mechanism of programmed cell death. (1) Hsu, H., J. Xiong, and D. V. Goeddel (1995) Cell 81, 495-504. (2) Chinnaiyan, A. M. et al. (1995) Cell 81, 505-512. (3) Harper, J. W. et al. (1993) Cell 75, 805-816.