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• W2084711835 abstract "nuclear envelope nuclear pore complex nucleoporin ribonucleoprotein nuclear localization signal Nucleocytoplasmic Transport and the Nuclear Pore ComplexUnlike their prokaryotic counterparts, eukaryotic cells separate the nuclear synthesis of DNA and RNA from cytoplasmic protein synthesis with a barrier termed the nuclear envelope (NE).1 The NE is perforated by large proteinaceous assemblies, called nuclear pore complexes (NPCs), which act as the sole gatekeepers controlling the exchange of material between the two locales (reviewed in Ref. 1Wente S.R. Science. 2000; 288: 1374-1377Crossref PubMed Scopus (215) Google Scholar). NPCs are freely permeable to small molecules (such as water and ions), but they restrict the movement of larger molecules (such as proteins and RNAs) across the NE. To overcome this barrier, macromolecules carry specific signals that allow them to access the nucleocytoplasmic transport machinery of the cell. In this way the cell ensures that only selected macromolecules can travel between the nucleus and cytoplasm (reviewed in Ref. 2Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1002) Google Scholar).Operationally, NPCs are composed of proteins called nucleoporins (or Nups) forming the stationary phase for nucleocytoplasmic exchange, whereas the mobile phase consists of soluble transport factors and their cargoes. As nucleocytoplasmic transport is driven by a series of specific interactions between components of both phases, it is frequently difficult to determine which proteins are permanent constituents of the NPC. Nevertheless, to understand how transport occurs, we must characterize the players in both phases and understand how their interplay leads to the coordinated vectorial exchange of macromolecules across the NE. In this review we focus on recent results that shed light on how some of these proteins interact to contribute to the elaborate NPC architecture and its function as a transport machine.Architecture of the NPCNPCs from different organisms share a common fundamental architecture (3Yang Q. Rout M.P. Akey C.W. Mol. Cell. 1998; 1: 223-234Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). These similarities likely provide clues as to the key features common to a functioning transport machine. The NPC is a large octagonally symmetric cylindrical structure. In yeast it estimated to be ∼50 MDa, whereas in metazoans it is over twice this mass (3Yang Q. Rout M.P. Akey C.W. Mol. Cell. 1998; 1: 223-234Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 4Reichelt R. Holzenburg A. Buhle Jr., E.L. Jarnik M. Engel A. Aebi U. J. Cell Biol. 1990; 110: 883-894Crossref PubMed Scopus (368) Google Scholar). Given that a ribosome at 4 MDa contains ∼80 proteins, it might be expected that the NPC would contain hundreds of different nucleoporins. However, it has recently been shown that the yeast NPC contains only ∼30 different proteins and the vertebrate NPC contains perhaps a few more (5Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar). This raises the question of how such a large complex can be constructed from so few component parts. The answer appears to lie in the symmetry of the structure (Figs.Figure 1, Figure 2, Figure 3). The NPC is comprised of a cylindrical core from which numerous peripheral filaments project toward the nucleus and cytoplasm (reviewed in Ref. 6Allen T.D. Cronshaw J.M. Bagley S. Kiseleva E. Goldberg M.W. J. Cell Sci. 2000; 113: 1651-1659Crossref PubMed Google Scholar) (Figs. 1 and2). The remarkable symmetry of the NPC is most apparent in the central core. Not only is it composed of eight identical spokes, but each spoke is also seemingly mirror symmetrical both in a plane parallel to the NE and in a perpendicular plane running through the cylindrical axis. As predicted from this symmetry, all nucleoporins examined thus far are present in multiple copies (apparently 1, 2, or 4 copies per spoke and hence 8, 16, or 32 copies per NPC), and most are localized to both the nuclear and cytoplasmic sides of the NE (5Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 6Allen T.D. Cronshaw J.M. Bagley S. Kiseleva E. Goldberg M.W. J. Cell Sci. 2000; 113: 1651-1659Crossref PubMed Google Scholar, 7Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (296) Google Scholar) (Fig. 3). By combining this symmetry with the relatively large size of most known nucleoporins (generally between 50 and 360 kDa), it becomes clear how the massive NPC can actually be constructed from a comparatively small number of proteins. Furthermore, the large size of nucleoporins potentially allows them to span between more than one domain of the NPC. However, for simplicity, we will begin by considering each major morphological NPC domain in turn and examine how they may combine to form the complete functional machine.Figure 2Visualization of NPC substructures.Scanning electron microscopy (left) of a vertebrate (Xenopus) NPC viewed en face from the cytoplasm best reveals the cytoplasmic filaments (CF); an NPC viewed similarly from the nucleoplasm shows the nuclear basket (NB). The structures of the central core are revealed by three-dimensional protein density maps generated by cryoelectron microscopy and image processing (CryoEM, right) of both vertebrate and yeast NPCs. The positions of the spoke (SP) and central transporter (T) are indicated on both the en face projection map (top row) and longitudinal slice (bottom row) of the vertebrate NPC. The positions of the cytoplasmic ring (CR), nuclear ring (NR), outer spoke-ring (OR), and inner spoke-ring (IR) are indicated on a longitudinal slice. Diagrams at the left of the micrographs show the corresponding orientation of the NPC. Micrographs were kindly provided by Martin Goldberg and Terry Allen (SEM) and Chris Akey (CryoEM).Bar, 50 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Increasing resolution maps of the NPC substructure. Immunoelectron microscopy (ImmunoEM) has begun to map the position of the nucleoporins within the NPC, whereas mass spectrometry (MS) is one of the new techniques being used to map the direct interactions between individual nucleoporins (5Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 14Rappsilber J. Siniossoglou S. Hurt E.C. Mann M. Anal. Chem. 2000; 72: 267-275Crossref PubMed Scopus (172) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Pore Membrane Domain and Formation of the Nuclear PoreThe nuclear envelope is composed of three biochemically distinct domains. The outer NE membrane is continuous with the endoplasmic reticulum and the inner membrane lies within the nucleus. Nuclear pores are created by a fusion of these two membranes, thus defining the third membrane domain, the pore membrane. The resulting channel connects the nucleoplasm with the cytoplasm, and integral membrane proteins localized to this domain are termed Poms (poremembrane proteins). Although surprisingly little is known about the function of each Pom, they likely play a central role in NPC assembly by initiating the formation of the pore membrane domain, stabilizing it, and serving as a membrane anchor site for the growing NPC. Remarkably, little homology has been found so far between Poms from different organisms, but as the mechanism of pore formation is probably conserved, it seems likely that such homologues exist and have yet to be identified.NPCs assemble continuously throughout interphase (8Maul G.G. Price J.W. Lieberman M.W. J. Cell Biol. 1971; 51: 405-418Crossref PubMed Scopus (125) Google Scholar, 9Winey M. Yarar D. Giddings Jr., T.H. Mastronarde D.N. Mol. Biol. Cell. 1997; 8: 2119-2132Crossref PubMed Scopus (170) Google Scholar); thus, the formation of the pore membrane domain must be fast and coincide with the insertion of the NPC, so that neither the nucleoplasm nor the ER lumen leaks during this process. Early assembly intermediates clearly have a pore membrane domain but apparently very little else and are presumably stabilized by integral pore membrane proteins such as Pom121p, which is recruited early in the reassembly process (10Gant T.M. Goldberg M.W. Allen T.D. Curr. Opin. Cell Biol. 1998; 10: 409-415Crossref PubMed Scopus (30) Google Scholar, 11Bodoor K. Shaikh S. Salina D. Raharjo W.H. Bastos R. Lohka M. Burke B. J. Cell Sci. 1999; 112: 2253-2264Crossref PubMed Google Scholar). Gp210, a pore membrane protein known to be a major constituent of the lumenal ring, is apparently recruited later in NPC assembly (11Bodoor K. Shaikh S. Salina D. Raharjo W.H. Bastos R. Lohka M. Burke B. J. Cell Sci. 1999; 112: 2253-2264Crossref PubMed Google Scholar). Gp210 is also hyperphosphorylated at the early stages of mitosis, and this modification may be important to initiate the mitotic disassembly of the NPC and nuclear envelope (12Favreau C. Worman H.J. Wozniak R.W. Frappier T. Courvalin J.C. Biochemistry. 1996; 35: 8035-8044Crossref PubMed Scopus (132) Google Scholar). Although Gp210 is a major protein in metazoan NPCs, the lack of an orthologue in yeast further suggests a possible role for Gp210 in NPC disassembly, as there is no NE disassembly step during yeast mitosis.The Central Core: The Spoke-Ring Complex and Central TransporterThe core of the NPC is considered to be the compact, highly symmetrical framework that underlies and stabilizes the central structure of the NPC. As might be expected, all the yeast nucleoporins that seem to fit within this category are relatively abundant components, localized to both faces of the NPC (5Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 7Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (296) Google Scholar). Surprisingly, only one-third of all the core nucleoporins is essential in yeast. This is likely a result of the symmetry and compact organization of the central core, such that proteins within this region make multiple contacts with each other and contribute to an interwoven framework that is stable to the loss of any individual component. This idea is supported by various genetic and biochemical data. One of the best examples for this connectivity is the well defined six-member Nup84p subcomplex (Fig. 3) (13Siniossoglou S. Lutzmann M. Santos-Rosa H. Leonard K. Mueller S. Aebi U. Hurt E. J. Cell Biol. 2000; 149: 41-54Crossref PubMed Scopus (143) Google Scholar). Most proteins in this complex make interactions with several of their neighbors, creating a network of protein interactions stabilizing the overall structure (14Rappsilber J. Siniossoglou S. Hurt E.C. Mann M. Anal. Chem. 2000; 72: 267-275Crossref PubMed Scopus (172) Google Scholar). That only two members of the complex are essential in yeast may reflect this stability, such that the complex can suffer loss of components without catastrophic consequences. When examined by electron microscopy, the complex has a Y-shaped morphology and a mass of ∼375 kDa. All the members are symmetrically disposed within each NPC and present in an estimated 16 copies (5Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 13Siniossoglou S. Lutzmann M. Santos-Rosa H. Leonard K. Mueller S. Aebi U. Hurt E. J. Cell Biol. 2000; 149: 41-54Crossref PubMed Scopus (143) Google Scholar). Thus, this one subcomplex alone could potentially account for ∼6 MDa of the 50-MDa yeast NPC! How this complex connects to the rest of the NPC still remains unclear, although it has been suggested that the arms of the “Y” structure interconnect to form one of the internal rings of the NPC (13Siniossoglou S. Lutzmann M. Santos-Rosa H. Leonard K. Mueller S. Aebi U. Hurt E. J. Cell Biol. 2000; 149: 41-54Crossref PubMed Scopus (143) Google Scholar).A detailed comparison of the core structure in vertebrates and yeast points to the presence of additional structures in vertebrates, including a radial arm and more elaborate nuclear and cytoplasmic rings (Fig. 2) (3Yang Q. Rout M.P. Akey C.W. Mol. Cell. 1998; 1: 223-234Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 7Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (296) Google Scholar, 15Akey C.W. Radermacher M. J. Cell Biol. 1993; 122: 1-19Crossref PubMed Scopus (335) Google Scholar). However, the features of the central transporter and spoke-ring complex are conserved between the two, as they are in all eukaryotes studied. At the molecular level, for known core components, there is also remarkable conservation. For example, mammalian Nup155 can functionally replace its orthologue, the yeast core protein Nup170p (16Aitchison J.D. Rout M.P. Marelli M. Blobel G. Wozniak R.W. J. Cell Biol. 1995; 131: 1133-1148Crossref PubMed Scopus (162) Google Scholar). The yeast Nup84p subcomplex also appears conserved; sequence comparisons suggest that most members of this subcomplex have metazoan orthologues, and a similar vertebrate complex can be isolated containing at least some mammalian counterparts of the yeast complex (17Fontoura B.M. Blobel G. Matunis M.J. J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (192) Google Scholar).How does the core contribute to NPC function? As all macromolecular transport across the NE occurs through the central transporter, supported within the core of the NPC, the core is obviously essential to transport. However, the core must also 1) maintain the structural integrity of the NPC as a barrier to diffusion while simultaneously being sufficiently flexible to withstand morphological changes in the nuclear envelope, 2) support the stepwise NPC assembly process, and 3) accommodate large transported cargo. Indeed, the spoke-ring complex and the central transporter have been observed in different morphological states by electron cryomicroscopy (15Akey C.W. Radermacher M. J. Cell Biol. 1993; 122: 1-19Crossref PubMed Scopus (335) Google Scholar, 18Akey C.W. Biophys. J. 1990; 58: 341-355Abstract Full Text PDF PubMed Scopus (125) Google Scholar). These different conformations suggest a sequential dilation of the central transporter, progressing from a resting state permeable to only smaller molecules to the triggering of a fully dilated state by the passage of the largest transport cargoes. Similar dramatic conformational changes within the central transporter and nuclear basket have been observed during the transport of large particles such as ribonucleoprotein particles (RNPs) (19Kiseleva E. Goldberg M.W. Daneholt B. Allen T.D. J. Mol. Biol. 1996; 260: 304-311Crossref PubMed Scopus (86) Google Scholar, 20Kiseleva E. Goldberg M. Allen T. Akey C. J. Cell Sci. 1998; 111: 223-236Crossref PubMed Google Scholar).Interestingly, in yeast, removal of the core components Nup170p and Nup188p increases the nonselective macromolecular permeability of the NPC (21Shulga N. Mosammaparast N. Wozniak R. Goldfarb D.S. J. Cell Biol. 2000; 149: 1027-1038Crossref PubMed Scopus (88) Google Scholar). These results are the first to define nucleoporins involved in controlling diffusion through the NPC and suggest that these proteins are either part of the transporter itself or anchor proteins that are.Nucleocytoplasmic Transport and the Peripheral NucleoporinsThe framework of the core also correctly positions the peripheral nucleoporins. These nucleoporins are considered accessible to the mobile phase of transport and thus play a more direct role in interacting with carriers and their cargoes. Cargoes destined for the nucleus carry a nuclear localization signal (NLS), whereas substrates to be exported from the nucleus harbor nuclear export sequence (reviewed in Ref. 2Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1002) Google Scholar). The signals are, in turn, recognized by a structurally related family of soluble transport receptor proteins collectively termed karyopherins (kaps; also known as importins, exportins, and transportins) (reviewed in Refs. 2Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1002) Google Scholar and 22Wozniak R.W. Rout M.P. Aitchison J.D. Trends Cell Biol. 1998; 8: 184-188Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Transport cargoes, such as nuclear proteins, messenger RNPs, tRNA, ribosomal proteins, ribosomal subunits, and small nuclear RNPs, have distinct NLSs or nuclear export sequences that are recognized by their own particular cognate transport factors. This interaction is controlled by the small GTPase Ran (see below and Refs. 23Cole C.N. Hammell C.M. Curr. Biol. 1998; 8: R368-R372Abstract Full Text Full Text PDF PubMed Google Scholar and 24Moore M.S. J. Biol. Chem. 1998; 273: 22857-22860Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Electron microscopy studies suggest that the karyopherin-NLS-cargo complex docks at multiple sites along the cytoplasmic filaments and through the NPC (25Akey C.W. Goldfarb D.S. J. Cell Biol. 1989; 109: 971-982Crossref PubMed Scopus (156) Google Scholar, 26Richardson W.D. Mills A.D. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1988; 52: 655-664Abstract Full Text PDF PubMed Scopus (372) Google Scholar). Thus, it is proposed that nuclear import is facilitated by a series of karyopherin docking and release steps, as the cargo-carrier complex moves along peripheral nucleoporins from the cytoplasmic filaments of the NPC through the central transporter, to the nucleoplasmic face, where the complex is released to the nuclear interior (27Blobel G. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 1-10Crossref PubMed Scopus (37) Google Scholar).FG Nucleoporins Provide an Abundance of Transport Factor Binding Sites at the NPCOf course, to fully understand how the NPC might directly contribute to transport, it is necessary to first characterize its components. The NPC is crammed with nucleoporins characterized by the presence of the FG dipeptide (Phe-Gly) repeat motifs. These repeats are present in nearly half the nucleoporins and often take the form of GLFG or FXFG repeats, separated by polar sequences of varying lengths. These so-called FG nucleoporins appear to be built upon the core structure and are present throughout the NPC, extending from the tips of the cytoplasmic filaments through the central transporter to the distal ring of the nuclear basket (Fig.3) (5Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 7Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (296) Google Scholar). As the FG nucleoporins are strategically positioned to be accessible to the mobile phase and interact directly with all of the karyopherins studied (as well as other cargo-carrying transport factors) (28Ryan K.J. Wente S.R. Curr. Opin. Cell Biol. 2000; 12: 361-371Crossref PubMed Scopus (211) Google Scholar), they are implicated directly in facilitating karyopherin/cargo movement across the NPC.Analysis of the structure of an FG repeat region bound to a karyopherin indicates that multiple FG repeats likely interact with numerous conserved hydrophobic pockets running along the outside of the karyopherin via phenylalanines in the FG repeat. Overall, the FG repeat region adopts an extended conformation with little intrinsic secondary structure. Furthermore, FG nucleoporins have been shown to form filaments (30Buss F. Kent H. Stewart M. Bailer S.M. Hanover J.A. J. Cell Sci. 1994; 107: 631-638Crossref PubMed Google Scholar) and colocalize with the filamentous structures of the NPC (reviewed in Refs. 6Allen T.D. Cronshaw J.M. Bagley S. Kiseleva E. Goldberg M.W. J. Cell Sci. 2000; 113: 1651-1659Crossref PubMed Google Scholar and 7Stoffler D. Fahrenkrog B. Aebi U. Curr. Opin. Cell Biol. 1999; 11: 391-401Crossref PubMed Scopus (296) Google Scholar). This is consistent with these proteins forming the majority of the filaments that emanate from the core and extend into the nucleoplasm and cytoplasm, although other possible conformational states cannot be excluded. As might be expected from their projection from the core of the NPC, in many cases FG nucleoporins are anchored to the core by one or the other of their ends (31Enarson P. Enarson M. Bastos R. Burke B. Chromosoma. 1998; 107: 228-236Crossref PubMed Scopus (42) Google Scholar, 32Del Priore V. Heath C. Snay C. MacMillan A. Gorsch L. Dagher S. Cole C. J. Cell Sci. 1997; 110: 2987-2999Crossref PubMed Google Scholar, 33Grandi P. Schlaich N. Tekotte H. Hurt E.C. EMBO J. 1995; 14: 76-87Crossref PubMed Scopus (133) Google Scholar, 34Schlaich N.L. Haner M. Lustig A. Aebi U. Hurt E.C. Mol. Biol. Cell. 1997; 8: 33-46Crossref PubMed Scopus (43) Google Scholar).Different Transport Factors, Different Docking SitesEvery transport factor studied can bind FG nucleoporins that have also been shown to bind other classes of transport factors (reviewed in Ref. 28Ryan K.J. Wente S.R. Curr. Opin. Cell Biol. 2000; 12: 361-371Crossref PubMed Scopus (211) Google Scholar). This fact and the observations that saturated or irreversible binding of some karyopherins to the NPC can be deleterious to other pathways suggest that pathways through the NPC overlap in specificity. However, considering the symmetry of the NPC and the abundance of FG nucleoporins there may be ∼160 transport factor binding sites per NPC. Although this provides a multitude of possible binding sites for each transport factor molecule, karyopherins have strong preferences for a restricted subset of FG nucleoporins (reviewed in Ref. 28Ryan K.J. Wente S.R. Curr. Opin. Cell Biol. 2000; 12: 361-371Crossref PubMed Scopus (211) Google Scholar). This could allow different karyopherins to simultaneously occupy different sites within a single NPC, while limiting the competitive interference between different pathways and increasing the potential transport flux in both directions. Indeed, it has been shown that a single NPC is capable of both exporting and importing different transport substrates (35Dworetzky S.I. Lanford R.E. Feldherr C.M. J. Cell Biol. 1988; 107: 1279-1287Crossref PubMed Scopus (200) Google Scholar). The NPC could also use such docking specificity as a way to globally regulate gene expression by simply modifying a nucleoporin dedicated to a particular nuclear transport factor. One of the most intriguing examples of this is the interaction between Kap121p and Nup53p in yeast. Although in vitro binding studies andin vivo fluorescence resonance energy transfer measurements demonstrate that Kap121p interacts with several different FG nucleoporins while transiting the NPC, both studies suggest that Nup53p is a specific docking site for Kap121p (36Marelli M. Aitchison J.D. Wozniak R.W. J. Cell Biol. 1998; 143: 1813-1830Crossref PubMed Scopus (133) Google Scholar, 37Damelin M. Silver P.A. Mol. Cell. 2000; 5: 133-140Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Thus, although not absolute, it appears that Nup53p could confer control over the Kap121p-mediated import pathway. Interestingly, Nup53p is phosphorylated at mitosis, and there is a concomitant decrease in the binding of the karyopherin Kap121p to the NPC although it remains to be determined if this results in a specific cell cycle-dependent change in nuclear import (36Marelli M. Aitchison J.D. Wozniak R.W. J. Cell Biol. 1998; 143: 1813-1830Crossref PubMed Scopus (133) Google Scholar).The Strategic Positioning of the Docking Sites: Efficient and Directional TransportFrom studies mapping the relative position of all the nucleoporins in yeast, it is striking that many FG nucleoporins are symmetrically disposed closely surrounding the central transporter, whereas FG nucleoporins localized exclusively to either the nucleoplasmic or cytoplasmic sides are placed further away from the core. This observation suggests that the directionality of transport factors through the NPC is conferred by the FG nucleoporins at the extremities of the NPC. It also likely that there are more subtle arrangements of docking sites within the symmetrical regions of the NPC in which precise order and distribution helps correctly direct transport factors as they transit the pore. Interestingly, like the core nucleoporins, complexes formed by the peripheral nucleoporins are also well conserved. Thus both the yeast Nsp1p nucleoporin subcomplex and the analogous vertebrate p62-p58-p54 complex are found on both sides of the NPC surrounding the central transporter (5Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 38Guan T. Muller S. Klier G. Pante N. Blevitt J.M. Haner M. Paschal B. Aebi U. Gerace L. Mol. Biol. Cell. 1995; 6: 1591-1603Crossref PubMed Scopus (134) Google Scholar, 39Grote M. Kubitscheck U. Reichelt R. Peters R. J. Cell Sci. 1995; 108: 2963-2972Crossref PubMed Google Scholar, 40Fahrenkrog B. Hurt E.C. Aebi U. Pante N. J. Cell Biol. 1998; 143: 577-588Crossref PubMed Scopus (93) Google Scholar). This together with the fact that there is only minimum amino acid sequence conservation in the repeat motifs between presumed orthologues from different species (41Fabre E. Hurt E. Annu. Rev. Genet. 1997; 31: 277-313Crossref PubMed Scopus (114) Google Scholar) suggests that the conservation lies in the functionality of the conserved binding sites themselves and in their similar strategic positions within the NPCs of different organisms.The Energetics of TransportIn addition to nucleoporins, sustained karyopherin-mediated nucleocytoplasmic exchange requires energy. The only known source of this energy is the small GTPase Ran (reviewed in Refs. 23Cole C.N. Hammell C.M. Curr. Biol. 1998; 8: R368-R372Abstract Full Text Full Text PDF PubMed Google Scholar and 24Moore M.S. J. Biol. Chem. 1998; 273: 22857-22860Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). However, as the translocation process itself is not linked to GTP hydrolysis, it is likely that the energy comes from a potential energy gradient across the NPC established by the maintenance of distinct pools of Ran: GTP-Ran in the nucleus and GDP-Ran in the cytoplasm (Fig. 4). This asymmetric distribution supports transport by triggering the assembly and disassembly of transport complexes in the correct compartments. Thus, importers release their cargoes when they interact with Ran-GTP in the nucleus, whereas exporters utilize Ran-GTP to bind their cargoes. Conversely, when the GTP on Ran is hydrolyzed (as is the case in the cytoplasm) importers can bind their cargoes, but exporters will release theirs.Figure 4The Ran cycle. Ran cycles between its GTP- and GDP-bound form dependent on its subcellular localization. The different forms of Ran confer directionality to transport by dictating where karyopherins bind and release their cargoes. See “The Energetics of Transport” for details. D, Ran-GDP;T, Ran-GTP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although Ran is soluble in both the nucleoplasm and cytoplasm, it is also directly tethered to the NPC through at least two different protein motifs within nucleoporins. The first domain is homologous to the cytoplasmic Ran-binding protein RanBP1. This domain binds both Ran-GTP and Ran-GDP and has been found in the cytoplasmic FG nucleoporin Nup358p (42Wu J. Matunis M.J. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). The second type of Ran binding domain, characterized by a zinc finger motif, binds Ran-GDP and is present on both Nup358p (43Yaseen N.R. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5516-5521Crossref PubMed Scopus (77) Google Scholar) and the nucleoplasmically disposed nucleoporin Nup153p (44Nakielny S. Shaikh S. Burke B. Dreyfuss G. EMBO J. 1999; 18: 1982-1995Crossref PubMed Scopus (181) Google Scholar). Ran binding to these distal nuclear and cytoplasmic components of the NPC may ensure a high concentration of Ran in the vicinity of the nuclear pore, improving the efficiency of the transport termination steps. This role may involve promoting the exchange of Ran between transport factors and maintaining the Ran-GTP/Ran-GDP gradient across the NPC. In addition, Nup358 tethers Ran-GAP (which activates the GTPase activity of Ran) to the NPC (45Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 46Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (376) Google Scholar). Localizing karyopherin docking sites, Ran binding sites, and Ran-GAP to the same nucleoporin may provide a means of ensuring highly efficient loading and unloading of transport factors and their cargoes during transport. This tethering" @default.
• W2084711835 created "2016-06-24" @default.
• W2084711835 creator A5003978275 @default.
• W2084711835 creator A5065548847 @default.
• W2084711835 date "2001-05-01" @default.
• W2084711835 modified "2023-09-27" @default.
• W2084711835 title "The Nuclear Pore Complex as a Transport Machine" @default.
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