Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties. Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties. Molecular exchange between the cytoplasm and nucleoplasm of cells is confined to pores in the envelope, which are formed and gated by a proteinaceous structure termed the nuclear pore complex (NPC) 1The abbreviations used are:NPCnuclear pore complexnupnucleoporinFG nupnucleoporin with FG motifsFG domainnup domain with FG motifskapkaryopherinMDmolecular dynamicsPONDRpredictors of natural disordered regionsAAamino acidCFPcyan fluorescent proteinmRNPmessenger ribonucleoprotein. 1The abbreviations used are:NPCnuclear pore complexnupnucleoporinFG nupnucleoporin with FG motifsFG domainnup domain with FG motifskapkaryopherinMDmolecular dynamicsPONDRpredictors of natural disordered regionsAAamino acidCFPcyan fluorescent proteinmRNPmessenger ribonucleoprotein. (1Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. The molecular architecture of the nuclear pore complex.Nature. 2007; 450: 695-701Crossref PubMed Scopus (812) Google Scholar, 2Beck M. Lucić V. Förster F. Baumeister W. Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electon tomography.Nature. 2007; 449: 611-615Crossref PubMed Scopus (283) Google Scholar). Metabolites and small proteins diffuse freely through the NPC (3Mohr D. Frey S. Fischer T. Güttler T. Görlich D. Characterisation of the passive permeability barrier of nuclear pore complexes.EMBO J. 2009; 28: 2541-2553Crossref PubMed Scopus (245) Google Scholar), but the diffusion of larger proteins and RNA is more selective and requires transport signals and mobile receptors termed karyopherins (kaps; importins, exportins, and transportins) (4Terry L.J. Shows E.B. Wente S.R. Crossing the nuclear envelope: Hierarchical regulation of nucleocytoplasmic transport.Science. 2007; 318: 1412-1416Crossref PubMed Scopus (410) Google Scholar). The molecular architecture of the NPC is similar in all eukaryotes examined. It features a ring-shaped scaffold that forms a central ∼50 nm transport conduit, eight short fibers extending from the scaffold into the cytoplasm, and a fibrous basket structure extending from the scaffold into the nucleoplasm (5Lim R.Y. Aebi U. Fahrenkrog B. Towards reconciling structure and function in the nuclear pore complex.Histochem. Cell Biol. 2008; 129: 105-116Crossref PubMed Scopus (105) Google Scholar, 6Frenkiel-Krispin D. Maco B. Aebi U. Medalia O. Structural analysis of a metazoan nuclear pore complex reveals a fused concentric ring architecture.J. Mol. Biol. 2010; 395: 578-586Crossref PubMed Scopus (83) Google Scholar). It also features a poorly defined structure in the center of the conduit (i.e. the transporter structure or central plug structure), which contains kap-cargo complexes in transit (2Beck M. Lucić V. Förster F. Baumeister W. Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electon tomography.Nature. 2007; 449: 611-615Crossref PubMed Scopus (283) Google Scholar, 7Akey C.W. Radermacher M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy.J. Cell Biol. 1993; 122: 1-19Crossref PubMed Scopus (335) Google Scholar, 8Yang Q. Rout M.P. Akey C.W. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications.Mol. Cell. 1998; 1: 223-234Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 9Fahrenkrog B. Aebi U. The nuclear pore complex: nucleocytoplasmic transport and beyond.Nat. Rev. Mol. Cell Biol. 2003; 4: 757-766Crossref PubMed Scopus (334) Google Scholar, 10Beck M. Förster F. Ecke M. Plitzko J.M. Melchior F. Gerisch G. Baumeister W. Medalia O. Nuclear pore complex structure and dynamics revealed by cryoelectron microscopy.Science. 2004; 306: 1387-1390Crossref PubMed Scopus (397) Google Scholar).Evidence suggests that the NPC passive diffusion conduit is juxtaposed on the facilitated transport conduit (3Mohr D. Frey S. Fischer T. Güttler T. Görlich D. Characterisation of the passive permeability barrier of nuclear pore complexes.EMBO J. 2009; 28: 2541-2553Crossref PubMed Scopus (245) Google Scholar, 11Feldherr C.M. Akin D. The location of the transport gate in the nuclear pore complex.J. Cell Sci. 1997; 110: 3065-3070Crossref PubMed Google Scholar, 12Keminer O. Peters R. Permeability of single nuclear pores.Biophys. J. 1999; 77: 217-228Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). This conduit must be flexible enough to accommodate kap-cargo complexes of different shapes and sizes while simultaneously maintaining a barrier against non-karyophilic proteins. Up to 60% of the channel capacity appears occluded at any given time by passing kap-cargo molecules (13Peters R. Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis.EMBO J. 1984; 3: 1831-1836Crossref PubMed Scopus (112) Google Scholar). The conduit may also be occluded by NPC components extending into the conduit. The NPC of yeast and mammals is composed of ∼30 proteins called nucleoporins (nups) in multiple copies for a total of ∼450 nups per NPC (14Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. The yeast nuclear pore complex: composition, architecture, and transport mechanism.J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 15Cronshaw J.M. Krutchinsky A.N. Zhang W. Chait B.T. Matunis M.J. Proteomic analysis of the mammalian nuclear pore complex.J. Cell Biol. 2002; 158: 915-927Crossref PubMed Scopus (766) Google Scholar). Half of these nups (the non-FG nups) have structures that resemble membrane coat proteins and together form a ring scaffold that functions as a "stent" to keep the pore membrane open (16Brohawn S.G. Leksa N.C. Spear E.D. Rajashankar K.R. Schwartz T.U. Structural evidence for common ancestry of the nuclear pore complex and vesicle coats.Science. 2008; 322: 1369-1373Crossref PubMed Scopus (166) Google Scholar, 17Hsia K.C. Stavropoulos P. Blobel G. Hoelz A. Architecture of a coat for the nuclear pore membrane.Cell. 2007; 131: 1313-1326Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). A few pore membrane nups have transmembrane domains and link the ring scaffold to the pore membrane (18Rexach M. Piecing together nuclear pore complex assembly during interphase.J. Cell Biol. 2009; 185: 377-379Crossref PubMed Scopus (9) Google Scholar, 19Hetzer M.W. Wente S.R. Border control at the nuclear pore complex: biogenesis and organization of the nuclear membrane and pore complexes.Dev. Cell. 2009; 17: 606-616Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 20Fernandez-Martinez J. Rout M.P. Nuclear pore complex biogenesis.Curr. Opin. Cell Biol. 2009; 21: 603-612Crossref PubMed Scopus (47) Google Scholar). The rest of the nups contain multiple copies of phenylalanine-glycine (FG) motifs dispersed over 150–700-amino acid (AA) domains that are intrinsically disordered (i.e. natively unfolded FG domains) (see Fig. 1) (21Denning D.P. Patel S.S. Uversky V. Fink A.L. Rexach M. Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2450-2455Crossref PubMed Scopus (368) Google Scholar). These disordered FG domains populate the transport conduit, but are anchored to the NPC ring scaffold by structured domains (22Denning D.P. Rexach M.F. Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins.Mol. Cell. Proteomics. 2007; 6: 272-282Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (see Fig. 1). Despite their structural disorder and functional redundancy, FG domains are essential for the survival of yeast (23Strawn L.A. Shen T. Shulga N. Goldfarb D.S. Wente S.R. Minimal nuclear pore complexes define FG repeat domains essential for transport.Nat. Cell Biol. 2004; 6: 197-206Crossref PubMed Scopus (299) Google Scholar) and presumably all eukaryotes.It is generally thought that a homogeneous network of random coils provided by intrinsically disordered FG domains forms the NPC permeability barrier (21Denning D.P. Patel S.S. Uversky V. Fink A.L. Rexach M. Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2450-2455Crossref PubMed Scopus (368) Google Scholar, 24Ribbeck K. Görlich D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.EMBO J. 2002; 21: 2664-2671Crossref PubMed Scopus (410) Google Scholar, 25Rout M.P. Aitchison J.D. Magnasco M.O. Chait B.T. Virtual gating and nuclear transport: the hole picture.Trends Cell Biol. 2003; 13: 622-628Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). In Aspergillus nidulans for example, a 5-min disruption of the NPC diffusion barrier coincides with the cell cycle-dependent dissociation of FG nups from the NPC (26De Souza C.P. Osmani A.H. Hashmi S.B. Osmani S.A. Partial nuclear pore complex disassembly during closed mitosis in Aspergillus nidulans.Curr. Biol. 2004; 14: 1973-1984Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Also, some yeast strains lacking nup FG domains have a compromised permeability barrier (27Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively-unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), although the effect is subtle and not always observed (23Strawn L.A. Shen T. Shulga N. Goldfarb D.S. Wente S.R. Minimal nuclear pore complexes define FG repeat domains essential for transport.Nat. Cell Biol. 2004; 6: 197-206Crossref PubMed Scopus (299) Google Scholar). Whereas in vivo analyses have been confounded by the functional redundancy of FG nups, reductionist approaches carried out in vitro with purified components have provided some insight. Indeed, the selective properties of the NPC toward kaps have been reconstituted in vitro using isolated FG domains on beads (27Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively-unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 28Allen N.P. Huang L. Burlingame A. Rexach M. Proteomic analysis of nucleoporin interacting proteins.J. Biol. Chem. 2001; 276: 29268-29274Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), FG domain hydrogels (29Frey S. Görlich D. FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties.EMBO J. 2009; 28: 2554-2567Crossref PubMed Scopus (91) Google Scholar), and FG domains attached to holes in membranes (30Jovanovic-Talisman T. Tetenbaum-Novatt J. McKenney A.S. Zilman A. Peters R. Rout M.P. Chait B.T. Artificial nanopores that mimic the transport selectivity of the nuclear pore complex.Nature. 2009; 457: 1023-1027Crossref PubMed Scopus (224) Google Scholar), highlighting the inherent capability of these domains to form selective diffusion barriers that can be specifically permeated by kaps. The exact configuration of FG domains within the NPC and the mechanism of kap movement across the NPC are the subject of much speculation (31Peters R. Translocation through the nuclear pore: Kaps pave the way.BioEssays. 2009; 31: 466-477Crossref PubMed Scopus (94) Google Scholar), but it seems clear that kaps and passing macromolecules must overcome a hydrophobic barrier imposed by FG domains (24Ribbeck K. Görlich D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.EMBO J. 2002; 21: 2664-2671Crossref PubMed Scopus (410) Google Scholar, 32Naim B. Zbaida D. Dagan S. Kapon R. Reich Z. Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier.EMBO J. 2009; 28: 2697-2705Crossref PubMed Scopus (58) Google Scholar, 33Jäkel S. Mingot J.M. Schwarzmaier P. Hartmann E. Görlich D. Importins fulfill a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains.EMBO J. 2002; 21: 377-386Crossref PubMed Scopus (216) Google Scholar).Elucidating the dynamic structure of individual FG domains and the intra- and intermolecular interactions they make is key to understanding NPC architecture. The FG domains represent ∼12% of the NPC mass, or 6.5 MDa of unresolved protein structure at the center of the NPC, controlling all nucleocytoplasmic traffic of macromolecules. The tertiary structure of only two FG domains has been characterized in detail, one from the vertebrate Nup153 and the other from yeast Nup116. The Nup153 FG domain adopts extended coil configurations that appear to compact upon binding a kap, giving rise to the proposal that kaps "collapse" the FG domain from extended to compact shapes to gain access across the NPC (34Lim R.Y. Fahrenkrog B. Köser J. Schwarz-Herion K. Deng J. Aebi U. Nanomechanical basis for selective gating by the nuclear pore complex.Science. 2007; 318: 640-643Crossref PubMed Scopus (235) Google Scholar). Consistently, the intrinsically disordered yeast nucleoporin Nup2 adopts extended coil configurations in purified form, but becomes more compact (i.e. to a smaller Stokes radius (RS)) upon kap binding (35Denning D.P. Uversky V. Patel S.S. Fink A.L. Rexach M. The Saccharomyces cerevisiae nucleoporin Nup2p is a natively unfolded protein.J. Biol. Chem. 2002; 277: 33447-33455Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In contrast, the Nup116 FG domain naturally adopts compact, collapsed coil configurations on average in the absence of kaps due in part to its intramolecular cohesion of coils mediated by FG motifs (36Krishnan V.V. Lau E.Y. Yamada J. Denning D.P. Patel S.S. Colvin M.E. Rexach M.F. Intramolecular cohesion of coils mediated by phenylalanine-glycine motifs in the natively unfolded domain of a nucleoporin.PLoS Comput. Biol. 2008; 4: e1000145Crossref PubMed Scopus (40) Google Scholar). Given these two seemingly disparate findings (collapsed coils or extended coils?), it became necessary to examine all FG nups to get a better picture of how their disordered structures are configured in their native state. For example, there are 11 FG nups in Saccharomyces cerevisiae (Fig. 1), and in principle, their FG domains could adopt any of several categories of intrinsically disordered structures such as molten globules, premolten globules, relaxed coils, or extended coils (37Dyson H.J. Wright P.E. Intrinsically unstructured proteins and their functions.Nat. Rev. Mol. Cell Biol. 2005; 6: 197-208Crossref PubMed Scopus (2984) Google Scholar, 38Uversky V.N. Protein folding revisited. A polypeptide chain at the folding-misfolding-non-folding crossroads: which way to go?.Cell. Mol. Life Sci. 2003; 60: 1852-1871Crossref PubMed Scopus (277) Google Scholar, 39Uversky V.N. Natively unfolded proteins: a point where biology waits for physics.Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1492) Google Scholar). These structures are distinguished from each other by their intramolecular packing density, which is defined by the molecular mass of the polypeptide chain and the hydrodynamic volume it occupies (40Tcherkasskaya O. Davidson E.A. Uversky V.N. Biophysical constraints for protein structure prediction.J. Proteome Res. 2003; 2: 37-42Crossref PubMed Scopus (49) Google Scholar). Currently, there are no computer-based structure prediction algorithms that differentiate between these different categories of disordered structures. Hence, we had to purify all FG domains from yeast nups to determine their Stokes radii to make structural assignments based on mass and hydrodynamic volume. In the process, we discovered that yeast FG nups are structurally heterogeneous and adopt distinct categories of disordered structures with separate functions in non-random distributions along their polypeptide chain. The implications of these findings to NPC architecture and function are discussed. Molecular exchange between the cytoplasm and nucleoplasm of cells is confined to pores in the envelope, which are formed and gated by a proteinaceous structure termed the nuclear pore complex (NPC) 1The abbreviations used are:NPCnuclear pore complexnupnucleoporinFG nupnucleoporin with FG motifsFG domainnup domain with FG motifskapkaryopherinMDmolecular dynamicsPONDRpredictors of natural disordered regionsAAamino acidCFPcyan fluorescent proteinmRNPmessenger ribonucleoprotein. 1The abbreviations used are:NPCnuclear pore complexnupnucleoporinFG nupnucleoporin with FG motifsFG domainnup domain with FG motifskapkaryopherinMDmolecular dynamicsPONDRpredictors of natural disordered regionsAAamino acidCFPcyan fluorescent proteinmRNPmessenger ribonucleoprotein. (1Alber F. Dokudovskaya S. Veenhoff L.M. Zhang W. Kipper J. Devos D. Suprapto A. Karni-Schmidt O. Williams R. Chait B.T. Sali A. Rout M.P. The molecular architecture of the nuclear pore complex.Nature. 2007; 450: 695-701Crossref PubMed Scopus (812) Google Scholar, 2Beck M. Lucić V. Förster F. Baumeister W. Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electon tomography.Nature. 2007; 449: 611-615Crossref PubMed Scopus (283) Google Scholar). Metabolites and small proteins diffuse freely through the NPC (3Mohr D. Frey S. Fischer T. Güttler T. Görlich D. Characterisation of the passive permeability barrier of nuclear pore complexes.EMBO J. 2009; 28: 2541-2553Crossref PubMed Scopus (245) Google Scholar), but the diffusion of larger proteins and RNA is more selective and requires transport signals and mobile receptors termed karyopherins (kaps; importins, exportins, and transportins) (4Terry L.J. Shows E.B. Wente S.R. Crossing the nuclear envelope: Hierarchical regulation of nucleocytoplasmic transport.Science. 2007; 318: 1412-1416Crossref PubMed Scopus (410) Google Scholar). The molecular architecture of the NPC is similar in all eukaryotes examined. It features a ring-shaped scaffold that forms a central ∼50 nm transport conduit, eight short fibers extending from the scaffold into the cytoplasm, and a fibrous basket structure extending from the scaffold into the nucleoplasm (5Lim R.Y. Aebi U. Fahrenkrog B. Towards reconciling structure and function in the nuclear pore complex.Histochem. Cell Biol. 2008; 129: 105-116Crossref PubMed Scopus (105) Google Scholar, 6Frenkiel-Krispin D. Maco B. Aebi U. Medalia O. Structural analysis of a metazoan nuclear pore complex reveals a fused concentric ring architecture.J. Mol. Biol. 2010; 395: 578-586Crossref PubMed Scopus (83) Google Scholar). It also features a poorly defined structure in the center of the conduit (i.e. the transporter structure or central plug structure), which contains kap-cargo complexes in transit (2Beck M. Lucić V. Förster F. Baumeister W. Medalia O. Snapshots of nuclear pore complexes in action captured by cryo-electon tomography.Nature. 2007; 449: 611-615Crossref PubMed Scopus (283) Google Scholar, 7Akey C.W. Radermacher M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy.J. Cell Biol. 1993; 122: 1-19Crossref PubMed Scopus (335) Google Scholar, 8Yang Q. Rout M.P. Akey C.W. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications.Mol. Cell. 1998; 1: 223-234Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 9Fahrenkrog B. Aebi U. The nuclear pore complex: nucleocytoplasmic transport and beyond.Nat. Rev. Mol. Cell Biol. 2003; 4: 757-766Crossref PubMed Scopus (334) Google Scholar, 10Beck M. Förster F. Ecke M. Plitzko J.M. Melchior F. Gerisch G. Baumeister W. Medalia O. Nuclear pore complex structure and dynamics revealed by cryoelectron microscopy.Science. 2004; 306: 1387-1390Crossref PubMed Scopus (397) Google Scholar). nuclear pore complex nucleoporin nucleoporin with FG motifs nup domain with FG motifs karyopherin molecular dynamics predictors of natural disordered regions amino acid cyan fluorescent protein messenger ribonucleoprotein. nuclear pore complex nucleoporin nucleoporin with FG motifs nup domain with FG motifs karyopherin molecular dynamics predictors of natural disordered regions amino acid cyan fluorescent protein messenger ribonucleoprotein. Evidence suggests that the NPC passive diffusion conduit is juxtaposed on the facilitated transport conduit (3Mohr D. Frey S. Fischer T. Güttler T. Görlich D. Characterisation of the passive permeability barrier of nuclear pore complexes.EMBO J. 2009; 28: 2541-2553Crossref PubMed Scopus (245) Google Scholar, 11Feldherr C.M. Akin D. The location of the transport gate in the nuclear pore complex.J. Cell Sci. 1997; 110: 3065-3070Crossref PubMed Google Scholar, 12Keminer O. Peters R. Permeability of single nuclear pores.Biophys. J. 1999; 77: 217-228Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). This conduit must be flexible enough to accommodate kap-cargo complexes of different shapes and sizes while simultaneously maintaining a barrier against non-karyophilic proteins. Up to 60% of the channel capacity appears occluded at any given time by passing kap-cargo molecules (13Peters R. Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis.EMBO J. 1984; 3: 1831-1836Crossref PubMed Scopus (112) Google Scholar). The conduit may also be occluded by NPC components extending into the conduit. The NPC of yeast and mammals is composed of ∼30 proteins called nucleoporins (nups) in multiple copies for a total of ∼450 nups per NPC (14Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. The yeast nuclear pore complex: composition, architecture, and transport mechanism.J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1143) Google Scholar, 15Cronshaw J.M. Krutchinsky A.N. Zhang W. Chait B.T. Matunis M.J. Proteomic analysis of the mammalian nuclear pore complex.J. Cell Biol. 2002; 158: 915-927Crossref PubMed Scopus (766) Google Scholar). Half of these nups (the non-FG nups) have structures that resemble membrane coat proteins and together form a ring scaffold that functions as a "stent" to keep the pore membrane open (16Brohawn S.G. Leksa N.C. Spear E.D. Rajashankar K.R. Schwartz T.U. Structural evidence for common ancestry of the nuclear pore complex and vesicle coats.Science. 2008; 322: 1369-1373Crossref PubMed Scopus (166) Google Scholar, 17Hsia K.C. Stavropoulos P. Blobel G. Hoelz A. Architecture of a coat for the nuclear pore membrane.Cell. 2007; 131: 1313-1326Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). A few pore membrane nups have transmembrane domains and link the ring scaffold to the pore membrane (18Rexach M. Piecing together nuclear pore complex assembly during interphase.J. Cell Biol. 2009; 185: 377-379Crossref PubMed Scopus (9) Google Scholar, 19Hetzer M.W. Wente S.R. Border control at the nuclear pore complex: biogenesis and organization of the nuclear membrane and pore complexes.Dev. Cell. 2009; 17: 606-616Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 20Fernandez-Martinez J. Rout M.P. Nuclear pore complex biogenesis.Curr. Opin. Cell Biol. 2009; 21: 603-612Crossref PubMed Scopus (47) Google Scholar). The rest of the nups contain multiple copies of phenylalanine-glycine (FG) motifs dispersed over 150–700-amino acid (AA) domains that are intrinsically disordered (i.e. natively unfolded FG domains) (see Fig. 1) (21Denning D.P. Patel S.S. Uversky V. Fink A.L. Rexach M. Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2450-2455Crossref PubMed Scopus (368) Google Scholar). These disordered FG domains populate the transport conduit, but are anchored to the NPC ring scaffold by structured domains (22Denning D.P. Rexach M.F. Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins.Mol. Cell. Proteomics. 2007; 6: 272-282Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (see Fig. 1). Despite their structural disorder and functional redundancy, FG domains are essential for the survival of yeast (23Strawn L.A. Shen T. Shulga N. Goldfarb D.S. Wente S.R. Minimal nuclear pore complexes define FG repeat domains essential for transport.Nat. Cell Biol. 2004; 6: 197-206Crossref PubMed Scopus (299) Google Scholar) and presumably all eukaryotes. It is generally thought that a homogeneous network of random coils provided by intrinsically disordered FG domains forms the NPC permeability barrier (21Denning D.P. Patel S.S. Uversky V. Fink A.L. Rexach M. Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2450-2455Crossref PubMed Scopus (368) Google Scholar, 24Ribbeck K. Görlich D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.EMBO J. 2002; 21: 2664-2671Crossref PubMed Scopus (410) Google Scholar, 25Rout M.P. Aitchison J.D. Magnasco M.O. Chait B.T. Virtual gating and nuclear transport: the hole picture.Trends Cell Biol. 2003; 13: 622-628Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). In Aspergillus nidulans for example, a 5-min disruption of the NPC diffusion barrier coincides with the cell cycle-dependent dissociation of FG nups from the NPC (26De Souza C.P. Osmani A.H. Hashmi S.B. Osmani S.A. Partial nuclear pore complex disassembly during closed mitosis in Aspergillus nidulans.Curr. Biol. 2004; 14: 1973-1984Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Also, some yeast strains lacking nup FG domains have a compromised permeability barrier (27Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively-unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), although the effect is subtle and not always observed (23Strawn L.A. Shen T. Shulga N. Goldfarb D.S. Wente S.R. Minimal nuclear pore complexes define FG repeat domains essential for transport.Nat. Cell Biol. 2004; 6: 197-206Crossref PubMed Scopus (299) Google Scholar). Whereas in vivo analyses have been confounded by the functional redundancy of FG nups, reductionist approaches carried out in vitro with purified components have provided some insight. Indeed, the selective properties of the NPC toward kaps have been reconstituted in vitro using isolated FG domains on beads (27Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Natively-unfolded nucleoporins gate protein diffusion across the nuclear pore complex.Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 28Allen N.P. Huang L. Burlingame A. Rexach M. Proteomic analysis of nucleoporin interacting proteins.J. Biol. Chem. 2001; 276: 29268-29274Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), FG domain hydrogels (29Frey S. Görlich D. FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties.EMBO J. 2009; 28: 2554-2567Crossref PubMed Scopus (91) Google Scholar), and FG domains attached to holes in membranes (30Jovanovic-Talisman T. Tetenbaum-Novatt J. McKenney A.S. Zilman A. Peters R. Rout M.P. Chait B.T. Artificial nanopores that mimic the transport selectivity of the nuclear pore complex.Nature. 2009; 457: 1023-1027Crossref PubMed Scopus (224) Google Scholar), highlighting the inherent capability of these domains to form selective diffusion barriers that can be specifically permeated by kaps. The exact configuration of FG domains within the NPC and the mechanism of kap movement across the NPC are the subject of much speculation (31Peters R. Translocation through the nuclear pore: Kaps pave the way.BioEssays. 2009; 31: 466-477Crossref PubMed Scopus (94) Google Scholar), but it seems clear that kaps and passing macromolecules must overcome a hydrophobic barrier imposed by FG domains (24Ribbeck K. Görlich D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.EMBO J. 2002; 21: 2664-2671Crossref PubMed Scopus (410) Google Scholar, 32Naim B. Zbaida D. Dagan S. Kapon R. Reich Z. Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier.EMBO J. 2009; 28: 2697-2705Crossref PubMed Scopus (58) Google Scholar, 33Jäkel S. Mingot J.M. Schwarzmaier P. Hartmann E. Görlich D. Importins fulfill a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains.EMBO J. 2002; 21: 377-386Crossref PubMed Scopus (216) Google Scholar). Elucidating the dynamic structure of individual FG domains and the intra- and intermolecular interactions they make is key to understanding NPC architecture. The FG domains represent ∼12% of the NPC mass, or 6.5 MDa of unresolved protein structure at the center of the NPC, controlling all nucleocytoplasmic traffic of macromolecules. The tertiary structure of only two FG domains has been characterized in detail, one from the vertebrate Nup153 and the other from yeast Nup116. The Nup153 FG domain adopts extended coil configurations that appear to compact upon binding a kap, giving rise to the proposal that kaps "collapse" the FG domain from extended to compact shapes to gain access across the NPC (34Lim R.Y. Fahrenkrog B. Köser J. Schwarz-Herion K. Deng J. Aebi U. Nanomechanical basis for selective gating by the nuclear pore complex.Science. 2007; 318: 640-643Crossref PubMed Scopus (235) Google Scholar). Consistently, the intrinsically disordered yeast nucleoporin Nup2 adopts extended coil configurations in purified form, but becomes more compact (i.e. to a smaller Stokes radius (RS)) upon kap binding (35Denning D.P. Uversky V. Patel S.S. Fink A.L. Rexach M. The Saccharomyces cerevisiae nucleoporin Nup2p is a natively unfolded protein.J. Biol. Chem. 2002; 277: 33447-33455Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In contrast, the Nup116 FG domain naturally adopts compact, collapsed coil configurations on average in the absence of kaps due in part to its intramolecular cohesion of coils mediated by FG motifs (36Krishnan V.V. Lau E.Y. Yamada J. Denning D.P. Patel S.S. Colvin M.E. Rexach M.F. Intramolecular cohesion of coils mediated by phenylalanine-glycine motifs in the natively unfolded domain of a nucleoporin.PLoS Comput. Biol. 2008; 4: e1000145Crossref PubMed Scopus (40) Google Scholar). Given these two seemingly disparate findings (collapsed coils or extended coils?), it became necessary to examine all FG nups to get a better picture of how their disordered structures are configured in their native state. For example, there are 11 FG nups in Saccharomyces cerevisiae (Fig. 1), and in principle, their FG domains could adopt any of several categories of intrinsically disordered structures such as molten globules, premolten globules, relaxed coils, or extended coils (37Dyson H.J. Wright P.E. Intrinsically unstructured proteins and their functions.Nat. Rev. Mol. Cell Biol. 2005; 6: 197-208Crossref PubMed Scopus (2984) Google Scholar, 38Uversky V.N. Protein folding revisited. A polypeptide chain at the folding-misfolding-non-folding crossroads: which way to go?.Cell. Mol. Life Sci. 2003; 60: 1852-1871Crossref PubMed Scopus (277) Google Scholar, 39Uversky V.N. Natively unfolded proteins: a point where biology waits for physics.Protein Sci. 2002; 11: 739-756Crossref PubMed Scopus (1492) Google Scholar). These structures are distinguished from each other by their intramolecular packing density, which is defined by the molecular mass of the polypeptide chain and the hydrodynamic volume it occupies (40Tcherkasskaya O. Davidson E.A. Uversky V.N. Biophysical constraints for protein structure prediction.J. Proteome Res. 2003; 2: 37-42Crossref PubMed Scopus (49) Google Scholar). Currently, there are no computer-based structure prediction algorithms that differentiate between these different categories of disordered structures. Hence, we had to purify all FG domains from yeast nups to determine their Stokes radii to make structural assignments based on mass and hydrodynamic volume. In the process, we discovered that yeast FG nups are structurally heterogeneous and adopt distinct categories of disordered structures with separate functions in non-random distributions along their polypeptide chain. The implications of these findings to NPC architecture and function are discussed. Supplementary Material Download .zip (16.58 MB) Help with zip files Download .zip (16.58 MB) Help with zip files
