A short region of its homeodomain is necessary for Engrailed nuclear export and secretion

Engrailed homeoprotein, a transcription factor involved in midbrain/hindbra in patterning, primarily localizes to the cell nucleus. However, significan t amounts of the protein are also found in the cell cytoplasm or associated with membrane microdomains enriched in cholesterol and glycosphingoglycoli pids (Joliot, A., Trembleau, A., Raposo, G., Calvet, S., Volovitch, M. and Prochiantz, A. (1997) Development 124, 1865-1875). This non-nuclear localiz ation, observed in vitro and in vivo, led us to investigate the possibility that Engrailed be transferred between nuclear and non-nuclear compartments . Monkey COS-7 cells expressing chick Engrailed-2 (cEN2) were fused with 3T 3 mouse fibroblasts and the passage of cEN2 from COS-7 to 3T3 nuclei was fo llowed in the interspecies heterokaryons. We find that, 10 minutes followin g cell fusion, cEN2 is detected in the 3T3 nuclei of 80% of the heterokaryo ns demonstrating rapid cEN2 nuclear export. Export from donor nuclei can be saturated and is strongly reduced after deletion of a 11 amino acid-long D elta 1 sequence present within a slightly larger domain that extends betwee n helices 2 and 3 of the homeodomain and shows strong similarities with leu cine-rich nuclear export signals (NES). This putative NES, when fused with a nuclear reporter protein, allows its nuclear export, demonstrating that i t is not only necessary but also sufficient for nuclear export and can ther efore be considered as a true nuclear export sequence. In an earlier report (Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitc h, M. and Prochiantz, A. (1998) Current Biology 8, 856-863), we demonstrate d that the Delta 1 sequence is necessary for the access of cEN2 to the lume n of a membrane compartment and for its intercellular transfer. The present study thus strongly suggests that the regulation of Engrailed nuclear expo rt could play a role not only in Engrailed transcriptional activity but als o in its ability to gain access to a secretory compartment.