A fraction of the yeast nucleoporin Nic96p is localized at the terminal ring of the nuclear basket. When Nic96p was affinity purified from glutaraldehyde-treated spheroplasts, it was found to be associated with Mlp2p. Mlp2p, together with Mlp1p, are the yeast Tpr homologues, which form the nuclear pore-attached intranuclear filaments (Strambio-de-Castillia, C., Blobel, G., and Rout, M. P. (1999) J. Cell Biol. 144, 839 -855). Double disruption mutants of MLP1 and MLP2 are viable and apparently not impaired in nucleocytoplasmic transport. However, overproduction of MLP1 causes nuclear accumulation of poly(A)؉ RNA in a chromatin-free area of the nucleus.Nuclear pore complexes are complex structures within the nuclear membrane, which mediate nuclear import and export of transport substrates and shuttling receptors (1). Each nuclear pore complex (NPC) 1 consists of a basic framework (i.e. the ring/spoke complex) which exhibits an 8-fold symmetry and spans the double nuclear membrane (2-4). The NPC is attached to peripheral structures such as the short cytoplasmic filaments, the nuclear basket, the nuclear envelope lattice, pore-attached intranuclear filaments, and the nuclear lamina (5-13). Recently, some of these peripheral elements of the NPC gained increased attention since they were proposed to play an important role in the initial docking step of the transport substrate to the pores and the final release from the pores (8, 14 -19). In particular, the large nucleoporin Nup358, located at the tips of the short cytoplasmatically attached NPC filaments, is thought to mediate one of the first contacts with transport cargos via the importin/karyopherin ␣/␤ complex (15, 19 -21). On the other side of the nuclear envelope, release of the import cargo from the nuclear pores appears to occur at the terminal ring of the nuclear basket, and Nup153 might be involved in this final process (16,18,22). However, electron microscopy revealed that NPCs do not abruptly end at the nuclear basket, but are connected to a complicated meshwork of intranuclear structures. These structures consist of pore-attached filaments, which deeply penetrate into the nuclear interior or an underlying nuclear envelope lattice (6,7,11,(23)(24)(25)(26). Therefore, it could be hypothesized that nuclear transport does not end after release of the import cargo from the nuclear basket and followed by intranuclear diffusion, but instead the facilitated transport through the pore could continue on intranuclear "tracks" to distinct intranuclear sites. In a similar way, export cargos may use intranuclear filaments for transport from the nuclear interior to the NPCs. Accordingly, the "gene gating hypothesis" was presented to propose that nuclear pores are connected via intranuclear tracks to distinct locations inside the nucleus (27).The molecular composition of the intranuclear filamentous system (often referred to as the "nuclear skeleton" or "nuclear matrix"), which has been visualized by a variety of electron microscopy techniques is still poorly characte...

This study aimed to investigate the association of the methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms with serum drug levels and toxicities after high-dose methotrexate (MTX) infusion. The study included 37 children with acute lymphoblastic leukemia or non-Hodgkin lymphoma. Serum MTX levels and toxicities of bone marrow, liver and kidney were analysed. Genotype analysis of the C677T and A1298C gene polymorphisms from genomic DNA of the subjects was performed by real-time PCR. Subjects with MTHFR polymorphism for C677T (CT, TT) had significantly higher MTX levels at 24 h (p = 0.009), and these genotypes did not seem to cause toxicity. Subjects with MTHFR polymorphism for A1298C (AC, CC) had significantly higher MTX levels at 48 h (p = 0.02), and had more grade III/IV anemia (p = 0.02), thrombocytopenia (p = 0.0001), elevated AST levels (p = 0.04) and frequent febrile neutropenic episodes (p = 0.004). The present study suggests that A1298C gene, but not C677T polymorphism is associated with MTX-related toxicity.

Human Nup93, the homologue of yeast Nic96p, is associated with a 205-kDa protein whose intracellular location and function is unknown. We show here that the yeast open reading frame YJL039c, which is homologous to this human p205, encodes the so far largest yeast nucleoporin. Accordingly, green fluorescent protein (GFP)-tagged YJL039c was localized to the nuclear pores and therefore named Nup192p. Affinity purification of ProtA-Nic96p from glutaraldehyde-fixed spheroplasts reveals association with Nup192p. NUP192 is essential for cell growth. A temperature-sensitive mutant nup192-15 is neither impaired in nuclear import of a SV40 nuclear localization sequence-containing reporter protein nor in mRNA export, but association of Nup49-GFP with nuclear pores is inhibited at the non-permissive temperature. By immunoelectron microscopy, Nup192p-ProtA is seen at the inner site of the nuclear pores, at a distance of 60 ؎ 15 nm from the central plane of the pore. This suggests that Nup192p is an evolutionarily conserved structural component of the nuclear pore complex with a preferential location at the inner site of the nuclear membrane.The nuclear pore complexes are huge structures embedded in the nuclear membrane that provide the major route for the passive diffusion of small molecules and active transport of large molecules between the nucleus and cytoplasm (1-4). In the electron microscope, the nuclear pore complex displays a modular organization, consisting of an octasymmetrical framework of eight spokes sandwiched between cytoplasmic and nuclear rings (5). The spokes embrace a central channel or "transporter" which gates nucleocytoplasmic transport in both directions. Attached to this core structure are peripheral elements, the cytoplasmic pore filaments, which extend from the cytoplasmic ring, and the nuclear basket attached to the nuclear ring and consisting of eight filaments that end in a distal ring (6 -9). Electron microscopy also showed that the 8-fold symmetry and modular aspects of pore complex organization have been conserved during evolution although yeast NPCs 1 are smaller as compared with Xenopus NPCs with respect to molecular mass and dimensions; in addition, some prominent structures present in vertebrates NPCs such as the luminal ring are absent in yeast (10, 11). The overall conservation in NPC morphology between yeast and vertebrates suggests that many components of the nuclear pore complex are conserved during evolution. Indeed, a significant number of NPC constituents are homologous between yeast and higher eukaryotes; however, often this homology is not easily noticed, and there are also cases in which no yeast homologue exists when a vertebrate Nup (e.g. Nup153) is known (12, 13). Conserved also is the machinery of soluble nucleocytoplasmic transport factors which interact with nucleoporins, in particular NPC constituents with FG/FXFG/GLFG repeat sequences (14). Accordingly, soluble transport factors such as importin/karyopherin ␣ and ␤ (Kap60p and Kap95p in yeast), the small GTPase Ran, and ...

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