Long lived
daf-2 mutant worms see a dramatic, up to 9-fold increase in the protein levels of F15E11.13 and F15E11.14 over the wild type (Dong et al. Science 2007). Both proteins belong to the same protein family of unknown function and each encoded by two genes (the protein products of F15E11.13 and Y19D10B.7 are the same, and the same is true for F15E11.14 and F15E11.1). The protein abundance increase of F15E11.13 and F15E11.14 is largely independent of the transcription factor
daf-16, which has been thought to mediate most or all the signaling from the insulin/IGF-1 receptor
daf-2. To our knowledge, this is the most robust
daf-2 dependent and yet
daf-16 independent regulation. When overexpressed together in the WT background, F15E11.13 and F15E11.14 confer resistance to heat and oxidative stress, but not longevity. However, their overexpression can further extend the lifespan of
daf-2(RNAi) and
daf-2(
e1370) mutants in a
daf-16 dependent manner. These results suggest that up-regulation of F15E11.13 and F15E11.14 promotes longevity, but it requires at least another effector of insulin signaling that likely resides downstream of
daf-16. To test whether F15E11.13 and F15E11.14 are necessary for the longevity of
daf-2 animals, we set out to delete all the genes encoding these proteins. F15E11.13/14/1, Y19D10B.7 and two other family members happen to be next to one another on Chromosome V, and we have found an almost ideal deletion in a WT C.elegans isolate JU258 that takes out all these genes. We have recombined this deletion into the N2 background and are in the process of removing the marker mutations used during recombination. The effect of this deletion on
daf-2 lifespan will be reported at the meeting. The C-terminal GFP fusion proteins of F15E11.13 and F15E11.14 expressed from their own promoters are both found in the cytoplasm and the nuclei of intestinal cells, body wall muscles and hypodermal cells. GFP-tagged F15E11.13 can immunoprecipitate F15E11.14 and vice versa. Recombinant F15E11.13 and F15E11.14 form a heterodimer as shown by crosslinking and gel-filtration experiments. The crystal structure of this heterodimer has been solved at 2.2 angstrom resolution. Based on the structural information, we are trying to find the biochemical activity of these proteins while designing better tags to look for their binding partners.