Molecules are transported between the cytoplasm and the nucleus through nuclear pore complexes (NPCs), massive proteinaceous structures that span the double membrane of the nuclear envelope (NE). Molecules smaller than ~20-40 kDa in size can passively diffuse through the NPC. However most protein, and nucleic acid is transported by receptor and energy dependent mechanisms (reviewed in [5–8]).
Nucleocytoplasmic transport is mediated by shuttling transport receptors termed karyopherins or importins/exportins (reviewed in [5, 7]). In the extensively studied classical nuclear import pathway, cargoes carrying a basic amino acid-rich nuclear localization sequence (NLS) bind to the adaptor importin a, which in turn associates with the import receptor importin β that mediates transport into the nucleus. A second class of import cargo directly binds to importin β in the absence of an adaptor. In the classical nuclear export pathway, cargoes carrying a leucine-rich nuclear export signal (NES) bind to the exportin CRM1 together with RanGTP to be transported out of the nucleus.
The small GTPase Ran, which binds directly to both importins and exportins, plays a key role in determining the directionality of nuclear transport. The GTP-bound form of Ran is concentrated in the nucleus and the GDP-bound form predominates in the cytoplasm, due to the nuclear localization of the Ran guanine nucleotide exchange factor RCC1 (RanGEF) and the cytoplasmic localization of the Ran GTPase- activating protein (RanGAP). The binding of RanGTP to karyopherins modulates the affinity of the receptors for cargo. When an importin-cargo complex encounters RanGTP in the nucleus, RanGTP promotes the dissociation of cargo from the receptor as well as dissociation of the importin from nucleoporins, and the importin-RanGTP complex is recycled back to the cytoplasm. The converse is true for exportins: intranuclear RanGTP promotes the binding of cargo to exportins, and when the RanGTP-containing export complex encounters RanGAP in the cytoplasm, GTP hydrolysis results in release of the cargo and regeneration of the free exportin [9–11].
The framework of the NPC consists of eight central spokes flanked by nuclear and cytoplasmic rings, forming a ring-spoke assembly that surrounds a central transport channel. Extending outward from the ring-spoke assembly are ~50-100-nm-long nuclear fibrils, which are joined in a basket-like structure ("nuclear basket"), and ~35-50-nm-long cytoplasmic fibrils (reviewed in [12, 13]). The NPC of both mammals and yeast comprise ~30 different nucleoporins, which are present at integral multiples of 8 copies, consistent with the 8-fold rotational symmetry of the NPC framework. Within the NPC, nucleoporins are typically organized in distinct subcomplexes that are localized to specific regions of the NPC . Approximately 1/3 of the nucleoporins contain multiple copies of the FG (phenylalanine-glycine) di-amino acid repeat. These FG repeats are clustered in domains ("FG domains") that are intrinsically unstructured. The FG domains appear to form the major diffusion barrier of the NPC [14, 15], and also serve as the key interaction sites for karyopherins during their transit through the NPC [12, 16].
In addition to undergoing reversible disassembly during mitosis in higher eukaryotes, NPCs are assembled throughout interphase in concert with NE growth . Moreover many nucleoporins have intranuclear pools that appear to undergo dynamic exchange with NPC localized forms . It is plausible that many if not most nucleoporins are imported into the nucleus by receptor-mediated pathways, but this process has not been studied in detail.
A conserved component of the NPC is the protein Tpr (for translocated promoter region)  and its homologs. Mammalian Tpr is a 267 kDa structurally bipartite protein comprising 2,349 amino acids. Its N-terminal 1,600 residue domain associates in a dimer to form a parallel two-stranded coiled-coil interrupted periodically along its length. The C-terminal domain comprising ~800 amino acids is highly acidic and is predicted to be unstructured . Tpr homologs have been characterized in Xenopus laevis , Saccharomyces cerevisiae (myosin-like proteins 1 and 2; Mlp1p and Mlp2p) , Drosophila melanogaster  and Arabidopsis thaliana . In mammalian cells, Tpr is localized to the nucleoplasmic fibrils of the NPC [1, 25] and is suggested to act as the main architectural element of the nuclear basket . Mammalian Tpr is tethered to the NPCs through interaction with Nup153 , whereas in yeast, Mlp1p and Mlp2p have been suggested to be anchored to the NPC by interactions with Nic96, or with Nup60 . Numerous functions have been attributed to vertebrate Tpr and its yeast homologs Mlp1p and Mlp2p in addition to a role in NPC architecture. These involve mRNA export [27–30], nuclear protein export [1, 31], silent telomeric chromatin organization and telomere length control [32–34], spindle pole assembly in yeast , unspliced RNA retention [4, 36] and localization and stabilization of a desumoylating enzyme Ulp1 [37, 38]. In addition, Drosophila Tpr has been linked to mitotic spindle organization and spindle checkpoint control .
In mammalian cells, classical nuclear protein import is not detectably affected in Tpr depleted cells [1, 40], whereas classical nuclear export is found to be significantly inhibited . Yeast cells carrying a double deletion of Mlp1p and Mlp2p display markedly slower import of a model cargo , but protein export has not been examined. A biochemical study conducted with Xenopus Laevis egg extracts demonstrates that importin β and importin α co-immunoprecipitate with Tpr . Whether this interaction is direct or indirect was not investigated, and its biological significance remains unresolved. To gain further insight into the role of Tpr in nucleocytoplasmic trafficking of protein, we investigated the interaction of Tpr with the karyopherins involved in classical nuclear import and export using quantitative binding assays. Our findings indicate that Tpr binds specifically and with relatively high affinity to these nuclear transport receptors, and support the notion that Tpr provides a docking site for importin α/β and CRM1 in nuclear import and export. Furthermore, the results of our binding analysis together with in vitro nuclear import assays indicate that the nuclear import of Tpr is efficiently mediated by the classical importin α/β pathway.