| During embryonic development, programs that regulate cell cycle progression, acquisition of cell fate, and cellular differentiation must be coordinated temporally and spatially throughout the embryo. The activity of molecules that regulate these processes must therefore be under particularly stringent control. One such molecule, Geminin (Gem), is a protein with roles both in DNA replication and in regulating cell fate during embryonic development. Prior studies demonstrated that Geminin performs multiple activities in the nucleus, and that at least one aspect of Geminin reactivation requires Geminin to localize to the nucleus. While Gem's distribution is predominantly nuclear in cycling cells, we observed that Gem protein transiently localizes to the cytoplasm during the differentiation of P19 mammalian embryonic carcinomal cells into neurons. Hence, we set out to determine the structural and mechanistic features underlying subcellular control of Geminin and their role in Geminin activity during embryonic development. We found that Geminin nuclear localization was dependent on a bipartite nuclear localization signal (NLS) located in the N-terminus of Xenopus Gem protein. Intriguingly, mutation or deletion of this motif revealed a second regulatory domain required to mediate Crm1-dependent export of Geminin. The bipartite NLS identified was the first structural motif to map to Geminin's N-terminal domain, which was originally shown to regulate neural cell fate acquisition. Therefore, we tested whether this activity was dependent on nuclear import. We noted that NLS-mutant Geminin was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. These data indicate that nuclear import of Geminin is required for its ability to regulate cell fate. Cross-species comparison of Geminin protein sequence revealed that, while the domain mediating Crm1-dependent export was well conserved at the sequence level, the bipartite NLS was present in non-mammalian vertebrates, but was not found in mammals. Instead, we determined that human Geminin employed an alternate N-terminal motif to regulate nuclear localization of Geminin. In summary, our data suggested that, rather than being constitutively nuclear, Geminin may undergo nucleocytoplasmic shuttling through distinct protein motifs and that this activity could affect changes in Geminin's subcellular localization accompanying differentiation. |
