The Nuclear Pore Complex: Oily Spaghetti or Gummy Bear?
2007; Cell Press; Volume: 130; Issue: 3 Linguagem: Inglês
10.1016/j.cell.2007.07.029
ISSN1097-4172
Autores Tópico(s)RNA Interference and Gene Delivery
ResumoIn this issue, Frey and Görlich, 2007Frey S. Görlich D. Cell. 2007; (this issue)Google Scholar provide new insight into the selective barrier that controls protein traffic through the nuclear pore complex. They show that a single protein domain of the nuclear pore protein Nsp1 can form a hydrogel that allows highly selective access of nuclear transport receptors and their cargos, but rejects other proteins of similar size. In this issue, Frey and Görlich, 2007Frey S. Görlich D. Cell. 2007; (this issue)Google Scholar provide new insight into the selective barrier that controls protein traffic through the nuclear pore complex. They show that a single protein domain of the nuclear pore protein Nsp1 can form a hydrogel that allows highly selective access of nuclear transport receptors and their cargos, but rejects other proteins of similar size. The nuclear pore complex (NPC) is a remarkable cellular machine. It is responsible for the exchange of proteins, RNAs, and other macromolecules between the cytoplasmic and nuclear compartments in eukaryotic cells, and it does so with extraordinary efficiency and specificity (Tran and Wente, 2006Tran E.J. Wente S.R. Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). For example, it is estimated that in humans every minute more than one kilogram of material is shuttled across all the NPCs in our body. Despite this staggering flow of mass, the NPC remains highly selective and allows the passage of molecules that are larger than 30–40 kDa only when bound to appropriate transport receptors. But how can a channel accommodate such high rates of transport without losing selectivity? New work by Frey and Görlich, 2007Frey S. Görlich D. Cell. 2007; (this issue)Google Scholar provides evidence that the nuclear pore may function akin to a gummy-like gel and demonstrates that a hydrogel assembled in vitro from a single nuclear pore protein domain is capable of mimicking some of the key permeability properties that were previously described for intact NPCs. With an estimated mass of 40–60 MDa, the NPC is among the largest macromolecular assemblies within the cell. Yet NPCs consist of only about 30 distinct proteins (nucleoporins or Nups), which are all present in multiple copies (Tran and Wente, 2006Tran E.J. Wente S.R. Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). The NPC channel is open for diffusion of small macromolecules, but beyond 30–40 kDa, nucleocytoplasmic transport substrates must contain specific targeting signals, generally referred to as nuclear localization signals (NLS) for nuclear import or nuclear export signals (NES) for nuclear export. These motifs are specifically recognized by soluble nuclear transport receptors that can traverse the NPC either alone or together with their bound cargo. The NPC is thought to be a passive sorting device whose task is to facilitate the selective diffusion of transport receptor-substrate complexes without imparting directionality. The free energy for nucleocytoplasmic transport is largely provided by a steep concentration gradient of the GTPase Ran. This molecule is highly enriched in the nucleus in its GTP-bound form, and GTP hydrolysis by Ran is directly coupled to the import/export cycle (Weis, 2003Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). Classes of nuclear pore proteins that contain phenylalanine-glycine (FG) repeats are likely to be critical for the process by which transport receptor-cargo complexes gain selective access to the NPC channel. Roughly one-third of all NPC proteins contain various FG repeat domains. FG repeats are highly unstructured or natively unfolded (Denning et al., 2003Denning D.P. Patel S.S. Uversky V. Fink A.L. Rexach M. Proc. Natl. Acad. Sci. USA. 2003; 100: 2450-2455Crossref PubMed Scopus (350) Google Scholar) and are thought to line the inner surface of the NPC channel (Tran and Wente, 2006Tran E.J. Wente S.R. Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, Weis, 2003Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). All known nuclear transport receptors can bind to FG-containing Nups, and interactions between transport receptors and FG repeats are essential for translocation through the NPC (Weis, 2003Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). However, the biophysical details of how these FG filaments contribute to the selective permeability of the NPC have been a matter of debate. Several models have been proposed to explain the gating behavior of the NPC (Figure 1). The "virtual gating" model views the NPC as a catalyst that can lower the activation energy for the translocation process (Rout et al., 2003Rout M.P. Aitchison J.D. Magnasco M.O. Chait B.T. Trends Cell Biol. 2003; 13: 622-628Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The barrier for large molecules to cross the NPC is normally high because they have to enter a narrow NPC tunnel, potentially crowded by FG repeats, leading to a significant decrease in their entropy. Binding of transport receptors to FG repeats would overcome this entropic barrier and would provide a significant kinetic advantage for cargos that are bound to transport receptors. Because the selectivity that is achieved by this mechanism does not rely on a mechanical barrier the term "virtual gating" was introduced to describe this model (Rout et al., 2003Rout M.P. Aitchison J.D. Magnasco M.O. Chait B.T. Trends Cell Biol. 2003; 13: 622-628Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). In a similar way, the "oily spaghetti" model suggests that long hydrophobic FG repeats normally occlude the NPC channel but can be pushed aside by receptor-cargo complexes (Macara, 2001Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref Scopus (716) Google Scholar). More recently, atomic force microscopy was used to show that certain FG repeats can indeed extend or collapse, suggesting that they act like polymer brushes that could contribute to an entropic barrier at the NPC (Lim et al., 2006Lim R.Y. Huang N.P. Koser J. Deng J. Lau K.H. Schwarz-Herion K. Fahrenkrog B. Aebi U. Proc. Natl. Acad. Sci. USA. 2006; 103: 9512-9517Crossref PubMed Scopus (199) Google Scholar). These kinetic models are in contrast to the "selective phase" model (Ribbeck and Görlich, 2001Ribbeck K. Görlich D. EMBO J. 2001; 20: 1320-1330Crossref PubMed Scopus (544) Google Scholar) that proposes the formation of a sieve-like meshwork within the NPC through interactions between FG-containing repeats (Figures 1B and 1C). Here, the size of the FG mesh determines the upper limits of the diffusion gate and mechanically restricts access of large molecules. However, the binding of transport receptors to FG repeats is proposed to dissolve the FG-FG network, and therefore transport receptors partition into this specific phase, which would explain how large receptor-cargo complexes gain exclusive access to the NPC. The selective phase model makes two key predictions. First, it requires that FG repeats form interactions, and second, it predicts the existence of a selective phase within the NPC channel. Interestingly, it was recently shown that the FG-repeat domain of the yeast nucleoporin Nsp1 can form a hydrogel-like structure in vitro that requires hydrophobic interactions between aromatic rings (Frey et al., 2006Frey S. Richter R.P. Görlich D. Science. 2006; 314: 815-817Crossref PubMed Scopus (392) Google Scholar). Furthermore, various FG-repeat domains can form weak and reversible homo- and heterotypic interactions in vitro and in vivo (Patel et al., 2007Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). However, interactions were only detected between FG domains that are located in the central channel of the NPC. This led to the proposal of a two-gate mechanism that combines a central gate, established by a "selective phase" of interacting FG repeats within the NPC channel and a "virtual gate," formed by noncohesive FG filaments located at the periphery of the NPC (Patel et al., 2007Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). These new studies firmly established that FG repeats can interact, but experimental evidence had been lacking for any of the proposed models to test whether the suggested mechanisms could provide sufficient selectivity to explain the permeability behavior of the NPC. In an astonishing set of experiments, Frey and Görlich now demonstrate that a hydrogel formed by the FG-repeat domain of Nsp1 displays selective properties that are reminiscent of the gating behavior of NPCs (Frey and Görlich, 2007Frey S. Görlich D. Cell. 2007; (this issue)Google Scholar). FG-hydrogels were assembled in vitro, and gel entry and diffusion rates of several proteins were examined by fluorescence microscopy. Remarkably, the influx of various nuclear transport receptors of the importin β family into the Nsp1 FG-hydrogel was ∼1000× faster than the entry of a control protein. Access of a model cargo bound to importin β was accelerated by more than 20,000× when compared to free cargo alone. Furthermore, the measurements of intra-gel diffusion rates matched up with recently published rates for NPC translocation derived from single molecule experiments (Kubitscheck et al., 2005Kubitscheck U. Grunwald D. Hoekstra A. Rohleder D. Kues T. Siebrasse J.P. Peters R. J. Cell Biol. 2005; 168: 233-243Crossref PubMed Scopus (190) Google Scholar, Yang et al., 2004Yang W. Gelles J. Musser S.M. Proc. Natl. Acad. Sci. USA. 2004; 101: 12887-12892Crossref PubMed Scopus (190) Google Scholar). Although these results are truly remarkable and highly suggestive, the question remains whether such a hydrogel exists within the NPC and whether these results reflect the in vivo physiology of nuclear transport. Interestingly, not every FG-hydrogel displays selectivity, and an Nsp1 FG-hydrogel that was formed according to previously published conditions (Frey et al., 2006Frey S. Richter R.P. Görlich D. Science. 2006; 314: 815-817Crossref PubMed Scopus (392) Google Scholar) allowed equal entry of all proteins and did not discriminate between nuclear transport receptors and cargo alone (Frey and Görlich, 2007Frey S. Görlich D. Cell. 2007; (this issue)Google Scholar). In order to achieve selective permeability, the total FG concentration within the gel had to be raised above 50 mM. This led the authors to introduce the concept of the saturated hydrogel, in which all the FG repeats have to extend completely and undergo a maximum number of interactions. This would allow the formation of a highly ordered mesh required to establish an efficient permeability barrier (Figure 1C). Despite the fact that the local concentration of FG repeats within the NPC may be high enough to achieve "saturation," it is hard to imagine how such a perfect FG network could be established in vivo, especially given that newly synthesized FG repeats would most likely immediately curl up and form intramolecular FG bridges. Indeed, in vitro gel formation can only be induced from lyophilized proteins under extreme pH and salt conditions. In order to overcome this conceptual problem, the authors suggest that nuclear transport receptors could act as chaperones. This would help to prevent intramolecular FG interactions after synthesis, with mesh formation being catalyzed once the nuclear pore protein reaches the NPC (Frey and Görlich, 2007Frey S. Görlich D. Cell. 2007; (this issue)Google Scholar). Saturated or not, the in vivo evidence for the existence of a FG-hydrogel within the NPC (Frey et al., 2006Frey S. Richter R.P. Görlich D. Science. 2006; 314: 815-817Crossref PubMed Scopus (392) Google Scholar) remains weak. Under more physiological conditions, the FG domain of Nsp1 formed neither homo- nor heterotypic interactions with other FG nucleoporins (Patel et al., 2007Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Therefore, the final answer to the question of whether the NPC looks more like a bowl of spaghetti or behaves like a gummy bear almost certainly requires additional structural and biophysical studies most likely paired with high-resolution single molecule experiments. A Saturated FG-Repeat Hydrogel Can Reproduce the Permeability Properties of Nuclear Pore ComplexesFrey et al.CellAugust 10, 2007In BriefThe permeability barrier of nuclear pore complexes (NPCs) controls the exchange between nucleus and cytoplasm. It suppresses the flux of inert macromolecules ≥ 30 kDa but allows rapid passage of even very large cargoes, provided these are bound to appropriate nuclear transport receptors. We show here that a saturated hydrogel formed by a single nucleoporin FG-repeat domain is sufficient to reproduce the permeability properties of NPCs. Importin β and related nuclear transport receptors entered such hydrogel >1000× faster than a similarly sized inert macromolecule. Full-Text PDF Open Archive
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