M.E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland
Running title: NPC becomes alive
* authors contributed equally
corresponding author: birthe.fahrenkrog@unibas.ch; phone: +41 61 267 2255; fax: +41 61 267 2109
Abstract
In this review we summarize the structure and function of the nuclear pore complex (NPC). Special emphasis is put on recent findings which reveal the NPC as a dynamic structure in the context of cellular events like nucleocytoplasmic transport, cell division and differentiation, stress response and apoptosis. Evidence for the involvement of nucleoporins in transcription and oncogenesis is discussed, and evolutionary strategies developed by viruses to cross the nuclear envelope are presented.
Keywords: nucleus; nuclear pore complex; nucleocytoplasmic transport; nuclear assembly; apoptosis; rheumatoid arthritis; primary biliary cirrhosis (PBC); systemic lupus erythematosus; cancer
Eukaryotic cells in interphase are characterized by distinct nuclear and cytoplasmic compartments separated by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the NE and they provide the sole gateways for molecular trafficking between the cytoplasm and nucleus of interphase eukaryotic cells. They allow passive diffusion of ions and small molecules, and facilitate receptor-mediated transport of signal bearing cargoes, such as proteins, RNAs and ribonucleoprotein (RNP) particles (Görlich and Kutay, 1999; Conti and Izaurralde, 2001; Macara, 2001; Fried and Kutay, 2003). A consensus model of the 3D architecture of the NPC has evolved from extensive electron microscopy (EM) and tomography studies in both yeast and higher eukaryotes (Unwin and Milligan, 1982; Hinshaw et al., 1992; Akey and Radermacher, 1993; Yang et al., 1998; Stoffler et al., 2003; Beck et al., 2004). Accordingly, the NPC consists of an eightfold symmetric central framework. The cytoplasmic ring moiety of the central framework is decorated with eight cytoplasmic filaments, whereas the nuclear ring moiety is topped with eight tenuous filaments that join distally into a massive distal ring and thereby form a distinct nuclear basket (Fig. 1). The overall 3D model of the NPC is conserved from yeast to higher eukaryotes, except for variations in linear dimensions among species (Fahrenkrog et al., 1998; Cohen et al., 2002; Stoffler et al., 2003).
Figure 1: 3D consensus model of the NPC. The main structural components of the NPC include the central framework embedded in NE membrane, the cytoplasmic ring moiety and cytoplasmic filaments, the nuclear ring moiety and the nuclear basket. Figure was modelled with a visual programming environment (ViPEr) at the Scripps Research Institute (http://www.scripps.edu). Reproduced, with permission (http://www.nature.com/nrm), from Fahrenkrog and Aebi (2003). |
Based on their molecular mass of ~125 MDa (Reichelt et al., 1990) and the high degree of 822 symmetry of their central framework, it is assumed that both vertebrate and yeast NPCs are composed of multiple copies of ~30 different proteins, called nucleoporins (Nups) (Panté and Aebi, 1996; Stoffler et al., 1999; Fahrenkrog and Aebi, 2002). Up to date, ~30 yeast (Table 1) and ~26 vertebrate (Table 2) nucleoporins have been identified and characterized (Rout et al., 2000; Cronshaw et al., 2002).
Table 1: Saccharomyces cerevisiae nucleoporins
aNup84
complex: C-Nup145, Nup120, Nup85, Nup84, Sec13p, Seh1p Abbreviations: D, Drosophila melanogaster; h, human; r, rat; Sp, Saccharomyces pombe; SPB, spindle pole body; X, Xenopus For more details see references: Stoffler et al. (1999); Rout et al. (2000); Fahrenkrog et al. (2001). |
Table 2: Vertebrate nucleoporins
aNup62
complex: Nup62, Nup58, Nup54, Nup45 Abbreviations: D, Drosophila melanogaster; Hv, Hydra vulgaris; r, rat; Sc, Saccharomyces cerevisiae; Sp, Saccaromyces pombe; AMLs, acute myeloid leukemias; PBC, primary biliary cirrhosis For more details se references: Stoffler et al. (1999); Fahrenkrog and Aebi (2002); Cronshaw et al. (2002). |
Many nucleoporins contain characteristic distinct domains of phenylalanine-glycine (FG) containing repeats. These repeat motifs are not required for targeting nucleoporins to the NPC but they play an important role in interactions between nucleoporins and soluble transport receptors (Table 1 and 2) (Rexach and Blobel, 1995). Recent experiments analyzing the role of FG repeats for nucleocytoplasmic transport have shown that the asymmetric localized FG repeats are dispensable, whereas certain symmetric ones are crucial for nucleocytoplasmic transport and cell viability (Strawn et al., 2004; Zeitler and Weis, 2004). In vitro pulldown assays employing Xenopus egg extracts, isolated rat liver nuclei or lysates from yeast nuclei demonstrate specific interactions between nucleoporins and the transport factors importin α, importin β and RanGTP (Iovine and Wente, 1997; Shah et al., 1998; Shah and Forbes, 1998; Marelli et al., 1998; Stochaj et al., 1998; Seedorf et al., 1999). FG repeats might function as docking sites for transport receptor molecules which in turn, interact with cargo. Additionally, the FG repeats might not only function as docking sites but also as parking sites to keep receptor-cargo complexes in a waiting position at the NPC for later subsequent passage through the central pore.
Precise localization of the nucleoporins in the NPC is essential for understanding their functions in nucleocytoplasmic transport and in NPC assembly at the molecular level. Immunolocalization of nucleoporins using immunogold EM has shown that most nucleoporins are symmetrically localized at both the cytoplasmic and nuclear periphery of the NPC and only few of them being located asymmetrically to either the cytoplasmic or nuclear periphery in both yeast and vertebrate NPC (Panté et al., 1994; Fahrenkrog et al., 1998; Rout et al., 2000; Fahrenkrog and Aebi, 2002; Cronshaw et al., 2002). Interestingly, many nucleoporins are organized into distinct subcomplexes within the NPC structure (Finlay et al., 1991; Panté et al., 1994; Belgareh et al., 2001; Walther et al., 2003). Some of these subcomplexes like the Nup107-160 complex and the Nup62 complex even stay intact during mitosis and might represent elementar building blocks of the NPC.
Despite the conserved structural features of NPCs among different species as yeast and vertebrates there have been some reports about cell-type specific expression of certain nucleoporins (Wang et al., 1994; Hu and Gerace, 1998; Nothwang et al., 1998; Cai et al., 2002; Coy et al., 2002; Olsson et al., 2004). For example, RanBP2L1, and POMFIL1 represent cell-type specific expressed proteins with homology to the nucleoporins RanBP2/Nup358 and POM121 which are coded by different genes (Wang et al., 1994; Nothwang et al., 1998; Cai et al., 2002; Coy et al., 2002). In contrast, the differentially expressed nucleoporins Nup45 and Nup58, both constituents of the Nup62 NPC subcomplex, seem to be generated by alternative splicing from the same gene (Hu and Gerace, 1998). The most recent report concerns the restricted expression of the transmembrane nucleoporin gp210: the mRNA as well as the expressed protein seem to be specific for cultured embryonic stem cells and certain polarized epithelial cells, whereas no expression could be detected in several cultured cell lines of fibroblastic and epithelial origin (Olsson et al., 2004). This result is of special importance since there seem to be only two transmembrane nucleoporins, gp210 and POM121, to anchor the entire 125 MDa NPC to the nuclear envelope, pointing towards a crucial role for POM121 in the architectural design of this multiprotein assembly.
3.1. Nucleoporin domain flexibility and nucleoporin turnover at the NPC
Contrary to the assumption that the NPC is a rather static structure, a number of recent studies have provided evidence that some of the nucleoporins are mobile within the NPC. For example, it has been shown that the vertebrate nucleoporin Nup98, originally localized to the nuclear basket (Radu et al., 1995), can also be found on the cytoplasmic face of the NPC (Griffis et al., 2003). Nup98 can dynamically associate with the nuclear pore and shuttle between the NPC and intranuclear bodies and additionally between the nucleus and the cytoplasm in a transcription-dependent manner (Zolotukhin and Felber, 1999; Griffis et al., 2002 and 2004). Because Nup98 plays a role in RNA export (Powers et al., 1997), its mobility proposes that Nup98 might associate with RNA close to its transcription site and then further accompany the processed RNA through the NPC into the cytoplasm.
Nup153 plays a role in the import of proteins into the nucleus as well as in the export of RNAs and proteins into the cytoplasm (Bastos et al., 1996; Shah and Forbes, 1998). Nup153 associated with export cargo shuttles between the nuclear and cytoplasmic faces of the NPC (Nakielny et al., 1999). Initially the vertebrate Nup153 had been localized to the distal ring of the nuclear basket (Panté et al., 1994) and close to or at the nuclear ring of the NPC (Walther et al., 2001). Immunogold EM with domain-specific antibodies against Nup153, in combination with recombinantly expressed epitope-tagged Nup153 in the Xenopus oocytes, has revealed a domain-specific topology within the NPC (Fahrenkrog et al., 2002). Nup153 seems to be anchored through its N-terminal domain to the nuclear ring moiety and its central Zn-finger domain to the distal ring of the NPC. By contrast, the C-terminal domain which harbors ~40 FG repeats (about 700 amino acids), appears to be highly mobile, as it has been localized throughout the nuclear basket and even at the cytoplasmic periphery of the central pore of the NPC (Fig. 2; Fahrenkrog et al. (2002 and 2004); Fahrenkrog and Aebi (2003)). In their native state FG-repeats are unfolded and highly unstructured (Bayliss et al., 2000; Allen et al., 2002; Denning et al., 2002 and 2003) so that the C-terminal domain of Nup153 could extend up to ~200 nm if completely stretched out, meaning that the C-terminal domain can reach all the way from the distal ring of the nuclear basket through the central pore into the cytoplasmic periphery of the NPC. This high mobility and structural flexibility of the FG repeats which act as binding/docking sites for cargo complexes might significantly increase the efficiency of cargo translocation through the NPC.
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Figure 2: Localization of the N-terminal, Zn-finger, and C-terminal domain of Nup153 in the 3D architecture of the NPC. Isolated Xenopus nuclei were preimmunolabeled
with the respective anti-Nup153-domain antibody directly conjugated
to 8-nm colloidal gold and prepared for EM by Epon embedding and thin-sectioning
(a) and by quick-freeze/freeze-drying/rotary metal-shadowing (b).
(a) Shown are a crosssectioned NE stretch with labeled NPCs, together with a gallery of selected examples of gold-labeled NPC in cross sections. c, cytoplasm; n, nucleus. Scale bars: 100 nm (b) Stereo images of selected examples of the nuclear face of X |
