Crystallization and preliminary X-ray diffraction analysis of human importin ß-Snail zinc finger domain complex.

Snail is a C2H2-type zinc finger transcriptional repressor that induces epithelial-mesenchymal transition by repression of E-cadherin expression levels during embryonic development and tumour progression. Snail is imported into the nucleus by importin ß through direct binding with its four zinc finger domain. The complex between importin ß and Snail four zinc finger domain was crystallized in order to understand the nuclear transport mechanism of Snail. The constituents of the complex were separately expressed and were then co-purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 228.2, b = 77.5, c = 72.0 Å, ß = 100.9° and diffracted to 2.5 Šresolution.

Crystallization of the flexible nuclear import receptor importin-beta in the unliganded state.

The transport of macromolecules across the nuclear envelope is an essential eukaryotic process that enables proteins such as transcription factors, polymerases and histones to gain access to the genetic material contained within the nucleus. Importin-beta plays a central role in the nucleocytoplasmic transport process, mediating nuclear import through a range of interactions with cytoplasmic, nuclear and nuclear pore proteins such as importin-alpha, Ran, nucleoporins and various cargo molecules. The unliganded form of the full-length yeast importin-beta has been expressed and crystallized. The crystals were obtained by vapour diffusion at pH 6.5 and 290 K. The crystals belonged to space group P2(1) (unit-cell parameters a = 58.17, b = 127.25, c = 68.52 A, beta = 102.23). One molecule is expected in the asymmetric unit. The crystals diffracted to 2.4 A resolution using a laboratory X-ray source and were suitable for crystal structure determination.

Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.

Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

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. CONCLUSION: 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.

The sal3(+) gene encodes an importin-beta implicated in the nuclear import of Cdc25 in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, the nuclear accumulation of Cdc25 peaks in G2 and is necessary for the proper timing of mitotic entry. Here, we identify the sal3(+) gene product as an importin-beta homolog that participates in the nuclear import of Cdc25. Loss of sal3(+) results in a cell cycle delay, failure to undergo G1 arrest under nitrogen-starvation conditions, and mislocalization of Cdc25 to the cytosol. Fusion of an exogenous classical nuclear localization sequence (cNLS) to Cdc25 restores its nuclear accumulation in a sal3 disruptant and suppresses the sal3 mutant phenotypes. In addition, we show that enhanced nuclear localization of Cdc25 at endogenous levels of expression advances the onset of mitosis. These results demonstrate that the nuclear translocation of Cdc25 is important for the timing of mitotic entry and that Sal3 plays an important role in this process.