CELL INTERNALIZATION OF THE 3RD HELIX OF THE ANTENNAPEDIA HOMEODOMAINIS RECEPTOR-INDEPENDENT

We have recently reported that a 16-amino acid long polypeptide corres ponding to the third helix of the DNA binding domain (homeodomain) of Antennapedia, a Drosophila transcription factor, is internalized by ce lls in culture (Derossi, D., Joliot, A. H., Chassaing, G., and Prochia ntz, A. (1994) J. Biol. Chem. 269, 10444-10450). The capture of the ho meodomain and of its third helix at temperatures below 10 degrees C ra ised the problem of the mechanism of internalization. The present demo nstration, that a reverse helix and a helix composed of D-enantiomers still translocate across biological membranes at 4 and 37 degrees C st rongly suggests that the third helix of the homeodomain is internalize d by a receptor-independent mechanism. The finding that introducing 1 or 3 prolines in the structure does not hamper internalization also de monstrates that the alpha-helical structure is not necessary. The data presented are compatible with a translocation process based on the es tablishment of direct interactions with the membrane phospholipids. Th e third helix of the homeodomain has been used successfully to address biologically active substances to the cytoplasm and nucleus of cells in culture (Theodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kub es, M., Jordan, P., Chneiweiss, H., Godement, P., and Prochiantz, A. ( 1995) J. Neurosci. 15, 7158-7167). Therefore, in addition to their phy siological implications (Prochiantz, A., and Theodore, L. (1995) BioEs says 17, 39-45), the present results open the way to the molecular des ign of cellular vectors.