ANTIGEN RELATED TO THE MAMMALIAN Alix/AIP1/Pdcd6ip IS CONSERVED IN SEA URCHINS.

The mammalian Alix/AIP1/Pdcd6ip/HP95 is an adaptor protein, first described for its capacity to bind to the calcium-binding protein ALG-2 and for its involvement in apoptosis (1). Recent evidence established that Alix regulates several cellular mechanisms, including endocytic membrane trafficking, multivesicular bodies biogenesis and cytoskeletal remodelling (2-5). Related proteins have been found in different metazoans. Alix regulates the signalling pathway involved in the oocyte maturation of X. laevis and plays a major role in motoneuron and in the imaginal eye discs differentiation of chicken and D. melanogaster embryos respectively; the related Alx-1 and Ego-2 seem to play a key role in the modulation of Notch-type signalling in C.elegans (6-9). Comparative analysis across phyla gives an invaluable contribution to the discovery of new gene functions. Due to the unravelled genome (10) and the key phylogenetic position, as sister group of the chordates within deuterostomes, the sea urchin model may shed new light in the role of the multifunctional Alix in fertilization, early development, cell specification and morphogenesis. By the comparison of mouse and human Alix with DNA/EST databases of S.purpuratus/P.lividus sea urchin species, we identified the putative Alix gene. The deduced amino acid sequence shows 46% identity and 63% conservation compared to the mammalian product, confirming that Alix is conserved in sea urchins. We performed Western blot and immunoprecipitation experiments with monoclonal antibodies against Alix and identified a single 100 kDa band in P.lividus eggs and embryos at early and late developmental stages. Preliminary assays of immunolocalization on whole mount eggs, showed that staining is confined to the cytosol. We plan to infer the function(s) that Alix may play in cell division and in signalling pathways involved in the specification of embryonic axes and the epithelial-mesenchyme transition.