Quantifying nucleoporin stoichiometry inside single nuclear pore complexes in vivo.

The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ~30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10-15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.

Nuclear pore complex protein sequences determine overall copolymer brush structure and function.

The transport of cargo across the nuclear membrane is highly selective and accomplished by a poorly understood mechanism involving hundreds of nucleoporins lining the inside of the nuclear pore complex (NPC). Currently, there is no clear picture of the overall structure formed by this collection of proteins within the pore, primarily due to their disordered nature. We perform coarse-grained simulations of both individual nucleoporins and grafted rings of nups mimicking the in vivo geometry of the NPC and supplement this with polymer brush modeling. Our results indicate that different regions or blocks of an individual NPC protein can have distinctly different forms of disorder and that this property appears to be a conserved functional feature. Furthermore, this block structure at the individual protein level is critical to the formation of a unique higher-order polymer brush architecture that can exist in distinct morphologies depending on the effective interaction energy between the phenylalanine glycine (FG) domains of different nups. Because the interactions between FG domains may be modulated by certain forms of transport factors, our results indicate that transitions between brush morphologies could play an important role in regulating transport across the NPC, suggesting novel forms of gated transport across membrane pores with wide biomimetic applicability.

Physical motif clustering within intrinsically disordered nucleoporin sequences reveals universal functional features.

Bioinformatics of disordered proteins is especially challenging given high mutation rates for homologous proteins and that functionality may not be strongly related to sequence. Here we have performed a novel bioinformatic analysis, based on the spatial clustering of physically relevant features such as binding motifs and charges within disordered proteins, on thousands of Nuclear Pore Complex (NPC) FG motif containing proteins (FG nups). The biophysical mechanism by which FG nups regulate nucleocytoplasmic transport has remained elusive. Our analysis revealed a set of highly conserved spatial features in the sequence structure of individual FG nups, such as the separation, localization, and ordering of FG motifs and charged residues along the protein chain. These functionally conserved features provide insight into the particular biophysical mechanisms responsible for regulation of nucleocytoplasmic traffic in the NPC, strongly constraining current models. Additionally this method allows us to identify potentially functionally analogous disordered proteins across distantly related species.

Prion formation by a yeast GLFG nucleoporin.

The self-assembly of proteins into higher order structures is both central to normal biology and a dominant force in disease. Certain glutamine/asparagine (Q/N)-rich proteins in the budding yeast Saccharomyces cerevisiae assemble into self-replicating amyloid-like protein polymers, or prions, that act as genetic elements in an entirely protein-based system of inheritance. The nuclear pore complex (NPC) contains multiple Q/N-rich proteins whose self-assembly has also been proposed to underlie structural and functional properties of the NPC. Here we show that an essential sequence feature of these proteins--repeating GLFG motifs--strongly promotes their self-assembly into amyloids with characteristics of prions. Furthermore, we demonstrate that Nup100 can form bona fide prions, thus establishing a previously undiscovered ability of yeast GLFG nucleoporins to adopt this conformational state in vivo.

A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins.

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.

Piecing together nuclear pore complex assembly during interphase.

All nucleocytoplasmic traffic of macromolecules occurs through nuclear pore complexes (NPCs), which function as stents in the nuclear envelope to keep nuclear pores open but gated. Three studies in this issue (Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Böttcher, and E. Hurt. 2009. J. Cell Biol. 185:387-395; Makio, T., L.H. Stanton, C.-C. Lin, D.S. Goldfarb, K. Weis, and R.W. Wozniak. 2009. J. Cell Biol. 185:459-491; Onishchenko, E., L.H. Stanton, A.S. Madrid, T. Kieselbach, and K. Weis. 2009. J. Cell Biol. 185:475-491) further our understanding of the NPC assembly process by reporting what happens when the supply lines of key proteins that provide a foundation for building these marvelous supramolecular structures are disrupted.

Intramolecular cohesion of coils mediated by phenylalanine--glycine motifs in the natively unfolded domain of a nucleoporin.

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine-glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin-cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.

Discovering novel interactions at the nuclear pore complex using bead halo: a rapid method for detecting molecular interactions of high and low affinity at equilibrium.

A highly sensitive, equilibrium-based binding assay termed "Bead Halo" was used here to identify and characterize interactions involving components of the nucleocytoplasmic transport machinery in eukaryotes. Bead Halo uncovered novel interactions between the importin Kap95 and the nucleoporins (nups) Nic96, Pom34, Gle1, Ndc1, Nup84, and Seh1, which likely occur during nuclear pore complex biogenesis. Bead Halo was also used to characterize the molecular determinants for binding between Kap95 and the family of nups that feature multiple phenylalanine-glycine motifs (FG nups). Binding was sensitive to the number of FG motifs present and to amino acid (AA) residues immediately flanking the FG motifs. Also, binding was reduced but not abolished when phenylalanine residues in all FG motifs were replaced by tyrosine or tryptophan. These results suggest flexibility in the binding pockets of Kap95 and synergism in binding FG motifs. The hypothesis that Nup53 and Nup59 bind directly to membranes through a C-terminal amphipathic alpha helix and to DNA via an RNA recognition motif domain was also tested and validated using Bead Halo. The results support a role for these nups in nuclear pore membrane biogenesis and in gene expression. Finally, Bead Halo detected binding of the nups Gle1, Nup60, and Nsp1 to phospholipid bilayers. This may reflect the known interaction between Gle1 and phosphoinositides and suggests similar interactions for Nup60 and Nsp1. As the Bead Halo assay detected molecular interactions in cell lysates, as well as between purified components, it can be adapted for large-scale proteomic studies using automated robotics and microscopy.

Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex.

Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.

Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins.

Nucleoporins with phenylalanine-glycine repeats (FG Nups) function at the nuclear pore complex (NPC) to facilitate nucleocytoplasmic transport. In Saccharomyces cerevisiae, each FG Nup contains a large natively unfolded domain that is punctuated by FG repeats. These FG repeats are surrounded by hydrophilic amino acids (AAs) common to disordered protein domains. Here we show that the FG domain of Nups from human, fly, worm, and other yeast species is also enriched in these disorder-associated AAs, indicating that structural disorder is a conserved feature of FG Nups and likely serves an important role in NPC function. Despite the conservation of AA composition, FG Nup sequences from different species show extensive divergence. A comparison of the AA substitution rates of proteins with syntenic orthologs in four Saccharomyces species revealed that FG Nups have evolved at twice the rate of average yeast proteins with most substitutions occurring in sequences between FG repeats. The rapid evolution of FG Nups is poorly explained by parameters known to influence AA substitution rate, such as protein expression level, interactivity, and essentiality; instead their rapid evolution may reflect an intrinsic permissiveness of natively unfolded structures to AA substitutions. The overall lack of AA sequence conservation in FG Nups is sharply contrasted by discrete stretches of conserved sequences. These conserved sequences highlight known karyopherin and nucleoporin binding sites as well as other uncharacterized sites that may have important structural and functional properties.

Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death.

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.

Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: I. How much of the data is theoretically interpretable by search engines?

An in-depth analysis of a multidimensional chromatography-mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight (QqTOF) geometry instrument was carried out. A total of 3269 CID spectra were acquired. Through manual verification of database search results and de novo interpretation of spectra 2368 spectra could be confidently determined as predicted tryptic peptides. A detailed analysis of the non-matching spectra was also carried out, highlighting what the non-matching spectra in a database search typically are composed of. The results of this comprehensive dataset study demonstrate that QqTOF instruments produce information-rich data of which a high percentage of the data is readily interpretable.

Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. New developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.

A thorough analysis of the protein interaction partners of the yeast GTPase Gsp1p was carried out by a multidimensional chromatography strategy of strong cation exchange fractionation of peptides followed by reverse phase LC-ESI-MSMS using a QSTAR instrument. This dataset was then analyzed using the latest developmental version of Protein Prospector. The Prospector search results were also compared with results from the search engine "Mascot" using a new results comparison program within Prospector named "SearchCompare." The results from this study demonstrate that the high quality data produced on a quadrupole selecting, quadrupole collision cell, time-of-flight (QqTOF) geometry instrument allows for confident assignment of the vast majority of interpretable spectra by current search engines.

Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin alpha in the nucleoplasm.

The yeast karyopherin heterodimer Kap60p.Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS). The alpha subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm. Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import. We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p.Gsp1p.GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p. Here we dissect the molecular mechanics of their KaRF activity. We show that Cse1p accelerates dissociation of Kap60p.NLS-cargo complexes and Kap60p.Nup2p complexes by increasing the affinity of Kap60p for its AIS motif. In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p.NLS complexes in a vectorial reaction mechanism. Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p.NLS-cargo complexes in the nucleoplasm of yeast. We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex.

A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex.

Karyopherins (Kaps) transport cargo across the nuclear pore complex (NPC) by interacting with nucleoporins that contain phenylalanine-glycine (FG) peptide repeats (FG Nups). As a test of the "affinity gradient" model for Kap translocation, we measured the apparent affinity of Kap95p to FG Nups representing three distinct regions of the S. cerevisiae NPC. We find that the affinity of Kap95p-Kap60p-cargo complexes to Nup1p (a nuclear basket Nup) is 225-fold higher than to Nup100p (a central scaffold Nup) and 4000-fold higher than to Nup42p (a cytoplasmic filament Nup), revealing a steep gradient of affinity for Kap95p complexes along the yeast NPC. A high affinity binding site for a Kap95p import complex was mapped to the C terminus of Nup1p, and, surprisingly, deletion of all FG repeats in that region did not eliminate binding of the complex. Instead, a 36-amino acid truncation of the C terminus of Nup1p reduced its affinity for the Kap95p import complex by 450-fold. Mutant yeast that express Nup1pDelta36 instead of full-length Nup1p display specific defects in Kap95p localization and Kap95p-mediated nuclear import. We conclude that a high affinity binding site for Kap95p at the nuclear basket increases the translocation efficiency of Kap95p import complexes across the NPC.

Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded.

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of "natively unfolded" proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.

The Saccharomyces cerevisiae nucleoporin Nup2p is a natively unfolded protein.

Little is known about the structure of the individual nucleoporins that form eukaryotic nuclear pore complexes (NPCs). We report here in vitro physical and structural characterizations of a full-length nucleoporin, the Saccharomyces cerevisiae protein Nup2p. Analyses of the Nup2p structure by far-UV circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, protease sensitivity, gel filtration, and sedimentation velocity experiments indicate that Nup2p is a "natively unfolded protein," belonging to a class of proteins that exhibit little secondary structure, high flexibility, and low compactness. Nup2p possesses a very large Stokes radius (79 A) in gel filtration columns, sediments slowly in sucrose gradients as a 2.9 S particle, and is highly sensitive to proteolytic digestion by proteinase K; these characteristics suggest a structure of low compactness and high flexibility. Spectral analyses (CD and FTIR spectroscopy) provide additional evidence that Nup2p contains extensive regions of structural disorder with comparatively small contributions of ordered secondary structure. We address the possible significance of natively unfolded nucleoporins in the mechanics of nucleocytoplasmic trafficking across NPCs.