Delmar VA et al. (2008), Xenopus importin beta validates human importin ...

XB-ART-37457

BMC Cell Biol 2008 Mar 22;9:14. doi: 10.1186/1471-2121-9-14.

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Xenopus importin beta validates human importin beta as a cell cycle negative regulator.

Delmar VA, Chan RC, Forbes DJ.

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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. 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. 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.

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Species referenced: Xenopus laevis
Genes referenced: kpnb1 ran

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	Figure 1. Xenopus importin beta shows close homology to human importin beta. The protein sequence of Xenopus importin beta shows very close homology to human importin beta with 94% identities (828/876, black boxes) and 97% positives (857/876 gray and black boxes). The amino acid composition, along with the length of the protein, is well conserved between Xenopus and human importin beta. Three of the conservative amino acid differences between the Xenopus and human importin beta sequence are at residues involved in FG-domain binding (F217Y [82–84], I265V [84], and L505V [84]).
	Figure 2. Xenopus importin beta is an authentic negative regulator of the fusion events in nuclear membrane formation. Addition of His-tagged Xenopus importin beta to a nuclear assembly reaction (+X-β) blocked nuclear membrane fusion, as shown by the lack of a solid nuclear rim stain by the green fluorescent membrane dye DHCC. The block to membrane fusion could be rescued by the addition of RanQ69L-GTP (+X-β +Ran). Where indicated, the added concentrations were 30 μM Xenopus importin beta and/or 40 μM RanQ69L-GTP. DNA was stained with DAPI. These observations are in accordance with experiments done with recombinant human importin beta in nuclear assembly reactions [9]. To better view the membranes, a section of the membrane stain (white dashed box) is enlarged by 3X (right panels). The bar represents 10 microns.
	Figure 3. Xenopus importin beta is an authentic negative regulator of nuclear pore assembly and is reversed by RanGTP. Pore-free BAPTA nuclear intermediates, which have fused nuclear membranes but contain no nuclear pores (left panel), when diluted into fresh cytosol (+ buffer), incorporate nuclear pores. The addition of His-tagged human importin beta (+h-β-Tag) or Xenopus untagged importin beta (+X-β) prevented nuclear pore assembly. Addition of RanQ69L-GTP with His-tagged human importin beta (+h-β-Tag +Ran) could not reverse the beta block to pore assembly, as previously observed [9]. However, addition of RanQ69L-GTP with untagged Xenopus importin beta (+X-β +Ran) did reverse the beta block to pore assembly. Nuclear pores were detected by the monoclonal antibody mAb414, which recognizes FG nucleoporins (FG Nups). Where indicated, importin beta was added at 20 μM and RanQ69L-GTP at 30 μM. The bar represents 10 microns. Black squares on the drawings at the right indicate FG-staining nuclear pores.
	Figure 4. Altering importin beta by addition of a His-tag renders importin beta insensitive to RanGTP specifically in its block to nuclear pore assembly. A. Pore-free BAPTA intermediates rescued in the presence of cytosol plus His-tagged Xenopus importin beta were not able to assemble nuclear pores (+Tag-X-β). When RanQ69L-GTP was added along with His-tagged Xenopus importin beta, the block to pore assembly could not be reversed (+Tag-X-β +Ran). Where indicated, importin beta was added at 10 μM and RanQ69L-GTP at 50 μM. The bar represents 10 microns. B. Pore-free BAPTA nuclear intermediates rescued in the presence of cytosol and untagged human or Xenopus importin beta were not able to assemble nuclear pores (+X-β or +h-β). The inhibitory concentration of 10 μM used here was determined to be the approximate minimum concentration for pore assembly inhibition in a separate experiment (data not shown). When RanQ69L-GTP was added along with untagged human importin beta, the block to pore assembly was partially reversed (+h-β +Ran). The Xenopus importin beta block was fully reversed (+X-β +Ran). To better visualize the FG-nucleoporin stain, a section of the images (white dashed box) was enlarged by 3X (right most panel). Where indicated, importin beta was added at 10 μM and RanQ69L-GTP at 50 μM. The bar represents 10 microns.

References [+] :

Albee, Phosphorylation of maskin by Aurora-A is regulated by RanGTP and importin beta. 2006, Pubmed, Xenbase