Xenopus importin beta validates human importin beta as a cell cycle negative regulator.
BACKGROUND
Human importin beta has been used in all Xenopus laevis in vitro nuclear assembly and spindle assembly studies. This disconnect between species raised the question for us as to whether importin beta was an authentic negative regulator of cell cycle events, or a dominant negative regulator due to a difference between the human and Xenopus importin beta sequences. No Xenopus importin beta gene was yet identified at the time of those studies. Thus, we first cloned, identified, and tested the Xenopus importin beta gene to address this important mechanistic difference. If human importin beta is an authentic negative regulator then we would expect human and Xenopus importin beta to have identical negative regulatory effects on nuclear membrane fusion and pore assembly. If human importin beta acts instead as a dominant negative mutant inhibitor, we should then see no inhibitory effect when we added the Xenopus homologue.


RESULTS
We found that Xenopus importin beta acts identically to its human counterpart. It negatively regulates both nuclear membrane fusion and pore assembly. Human importin beta inhibition was previously found to be reversible by Ran for mitotic spindle assembly and nuclear membrane fusion, but not nuclear pore assembly. During the present study, we observed that this differing reversibility varied depending on the presence or absence of a tag on importin beta. Indeed, when untagged importin beta, either human or Xenopus, was used, inhibition of nuclear pore assembly proved to be Ran-reversible.


CONCLUSIONS
We conclude that importin beta, human or Xenopus, is an authentic negative regulator of nuclear assembly and, presumably, spindle assembly. A difference in the Ran sensitivity between tagged and untagged importin beta in pore assembly gives us mechanistic insight into nuclear pore formation.
Delmar
Valerie A
VA
Section of Cell and Developmental Biology, Division of Biological Sciences 0347, University of California - San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0347, USA. vadelmar@yahoo.com
Chan
Rene C
RC
Forbes
Douglass J
DJ
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015536:Down-Regulation / Q000235:genetics; D006801:Humans; D008561:Membrane Fusion / Q000235:genetics; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000235:genetics / Q000378:metabolism; D022022:Nuclear Pore / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D029867:Xenopus Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein