Nucleoporins as Components of the Nuclear Pore Complex Core Structure and Tpr as the Architectural Element of the Nuclear Basket

Abstract

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

Vertebrate Nup53 Interacts with the Nuclear Lamina and Is Required for the Assembly of a Nup93-containing Complex

“…As a complement to these studies, we also examined the effects of depleting Nup93 on levels of other members of this interacting group. As shown in Figure 7, treatment of HeLa cells with Nup93-specific siRNAs reduced cellular levels of Nup93 and, as previously shown, the mAb414-reactive nups (Figure 7, A and B; Krull et al, 2004). Similarly, depleting Nup93 reduced cellular levels of Nup53, Nup155, and Nup205, but did not affect levels of Nup107, gp210, Mad1, or lamin B (Figure 7).…”

“…This conclusion is based both on the localization of Nup93 and Nup205 as discussed above (Krull et al, 2004) and on the tight association between Nup53 and the NE membrane. We have shown, using various urea extraction conditions, that the release of Nup53 from the NE membrane appears to largely parallel that of the lamins, requiring significantly higher concentrations of urea for extraction from the membrane than other nups.…”

“…Smythe et al (2000) have suggested that Xenopus Nup153 binds to lamin B 3 . Nup153 is located on the nuclear side of the NPC (Cordes et al, 1993;Sukegawa and Blobel, 1993) and it has been suggested that it links the core structures to the nuclear basket and the nuclear filamentous protein Tpr (Krull et al, 2004). These data place Nup153 in a separate subcomplex from the core associated Nup53.…”

“…Recent immunoelectron microscopy data from Krull et al (2004) suggest that both Nup93 and Nup205 are centrally positioned within the NPC near the pore membrane. Also consistent with this idea, Nup155 is symmetrically distributed on both faces of the NPC (Radu et al, 1993).…”

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

“…Different groups of Nups and their respective subcomplexes contribute to distinct structural components of the NPC. For example, Nup214/Nup84 are components of the cytoplasmic fibrils (Kraemer et al, 1994;Bastos et al, 1997), the Nup53/Nup155 complex is part of the central core (Marelli et al, 1998), and Nup153 and Tpr contribute to the nucleoplasmic ring and fibrils (Krull et al, 2004).When metazoan cells enter mitosis, their NE is disassembled allowing for the mitotic spindle to access the condensed chromatin. Several models based on previous findings have been proposed to explain the mechanism by which this occurs (reviewed in Hetzer et al, 2005).…”