Rabbit Anti-Human NUP88 (a.a. 551-650) Polyclonal antibody

Nucleoporins mediate transport of macromolecules across the nuclear pore complex, yet the function of many individual nucleoporins is largely unresolved. To address this question, we depleted cells of the cytoplasmic nucleoporins Nup214/CAN and Nup358/RanBP2 by RNA interference. Depletion of Nup214 resulted in codepletion of its binding partner, Nup88. Nuclear pore complexes assembled in the absence of Nup214/Nup88 or Nup358 were fully functional in nuclear protein import, whereas nuclear mRNA export was slightly impaired. Depletion of Nup358 had only a minor effect on nuclear protein export. In contrast, depletion of Nup214/Nup88 led to strongly reduced CRM1-mediated export of the shuttling transcription factor NFAT as well as a human immunodeficiency virus-Rev derivative. A specific role of Nup214 in protein export is furthered by the biochemical properties of a high-affinity complex containing Nup214, CRM1, RanGTP, and an export cargo. Our results show that the Nup214/Nup88 complex is required for efficient CRM1-mediated transport, supporting a model involving a high-affinity binding site for CRM1 at Nup214 in the terminal steps of export.

Izumi Y, Li J, Villers C, Hashimoto K, Burg MB, Ferraris JD. Mutations that reduce its specific DNA binding inhibit high NaCl-induced nuclear localization of the osmoprotective transcription factor NFAT5. Am J Physiol Cell Physiol 303: C1061-C1069, 2012. First published September 19, 2012; doi:10.1152/ajpcell.00265.2012.The transcription factor nuclear factor of activated T cell 5 (NFAT5) is activated by the stress of hypertonicity (e.g., high NaCl). Increased expression of NFAT5 target genes causes accumulation of protective organic osmolytes and heat shock proteins. Under normotonic conditions (similar to 300 mosmol/kgH(2)O), NFAT5 is distributed between the nucleus and cytoplasm, hypertonicity causes it to translocate into the nucleus, and hypotonicity causes it to translocate into the cytoplasm. The mechanism of translocation is complex and not completely understood. NFAT5-T298 is a known contact site of NFAT5 with its specific DNA element [ osmotic response element (ORE)]. In the present study, we find that mutation of NFAT5-T298 to alanine or aspartic acid not only reduces binding of NFAT5 to OREs (EMSA) but also proportionately reduces high NaCl-induced nuclear translocation of NFAT5. Combined mutation of other NFAT5 DNA contact sites (R293A/E299A/R302A) also greatly reduces both specific DNA binding and nuclear localization of NFAT5. NFAT5-T298 is a potential phosphorylation site, but, using protein mass spectrometry, we do not find phosphorylation at NFAT5-T298. Further, decreased high NaCl-induced nuclear localization of NFAT5 mutated at T298 does not involve previously known regulatory mechanisms, including hypotonicity-induced export of NFAT5, regulated by phosphorylation of NFAT5-S155, XPO1 (CRM1/exportin1)-mediated export of NFAT5 from the nucleus, or hypertonicity-induced elevation of NUP88, which enhances nuclear localization of NFAT5. We conclude that specific DNA binding of NFAT5 contributes to its nuclear localization, by mechanisms, as yet undetermined, but independent of ones previously described to regulate NFAT5 distribution.