UC San Diego

In eukaryotic cells, proteins known as karyopherins function during interphase as nuclear import and export receptors. Importin [beta] and Transportin, two such karyopherins, act to carry proteins with different recognition sites into the interphase nucleus. Once inside the nucleus, these karyopherins exchange their cargoes for RanGTP, which is exclusively nucleoplasmic. Interestingly, Importin [beta] has a very different role in mitosis. There it acts as a spatial regulator of mitotic events, including spindle assembly. Importin [beta] masks spindle assembly factors everywhere except in the vicinity of chromatin, where a high concentration of RanGTP exists. Recently, our lab demonstrated that Transportin also regulates mitotic events. However, the mechanism for this was not explored. Firm in the knowledge of Importin [beta]'s regulation of mitotic events, there are two obvious models for Transportin's regulation of spindle assembly. One is that Transportin acts by binding to and titrating RanGTP, thus modulating the release of spindle assembly factors by Importin [beta]. A second is that, like Importin [beta], Transportin functions directly in spindle assembly, itself binding and masking a set of spindle assembly factors. We employed different tools to test these mechanisms : M9M, a known inhibitor of Transportin in nuclear import, and TLB, a Transportin mutant with a truncated H8 loop that is desensitized to Ran-mediated cargo release. We used these molecular tools both in an in vitro extract derived from Xenopus laevis eggs and in an in vivo system, HeLa cells. Our results support the hypothesis that Transportin acts during mitosis, not indirectly through the titration of RanGTP, but directly by regulating spindle assembly factors. Potential factors will be discussed