Subcellular translocation signals regulate Geminin activity during embryonic development.
BACKGROUND
Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development.


RESULTS
We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem.


CONCLUSIONS
We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.
Boos
Aline
A
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Lee
Amy
A
Thompson
Dominic M
DM
Kroll
Kristen L
KL
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004475:Ectoderm / Q000166:cytology; D047108:Embryonic Development; D064248:Geminin; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D015398:Signal Transduction; D014981:Xenopus / Q000196:embryology / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism; D000097606:Exportin 1 Protein
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D064248:Geminin; D028884:Karyopherins; D019913:Nuclear Localization Signals; D018160:Receptors, Cytoplasmic and Nuclear; D029867:Xenopus Proteins; D000097606:Exportin 1 Protein; C406819:Cdt1 protein, Xenopus; C577998:GMNN protein, Xenopus