The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.     

Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p,Nup59p,and Nup170p

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.     

Importin beta contains a COOH-terminal nucleoporin binding region important for nuclear transport

Proteins containing a classical NLS are transported into the nucleus by the import receptor importin beta, which binds to cargoes via the adaptor importin alpha. The import complex is translocated through the nuclear pore complex by interactions of importin beta with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin beta. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin beta to a similar extent ( approximately 50%). An importin beta mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin beta possesses two nucleoporin binding sites, both of which are important for its nuclear import function.     

Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex.

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.     

Nup53p is a target of two mitotic kinases, Cdk1p and Hrr25p

Nuclear pore complexes (NPCs) form channels across the nuclear envelope and provide the sole sites of molecular exchange between the cytoplasm and nucleoplasm. The NPC is a target of a number of post-translational modifications, including phosphorylation, yet the functions of these modifications are ill defined. Here, we have investigated the mitotic specific phosphorylation of a yeast nucleoporin Nup53p. Two kinases were identified that phosphorylate Nup53p: the mitotic kinase Cdk1p/Cdc2p/Cdc28p and the casein kinase Hrr25p. Hrr25p was identified by screening 119 yeast kinases for their ability to phosphorylate Nup53p in vitro. Conditional alleles of Hrr25p support the conclusion that Hrr25p phosphorylates Nup53p in vivo. We further demonstrated using solution binding and affinity purification assays, that Hrr25p directly binds Nup53p in an interaction that is destabilized by the phosphorylation of Nup53p. Consistent with this observation, we observed that Hrr25p moves between distinct locations in the cell during the cell cycle including the nucleus, the cortex of the emerging bud and the spindle pole bodies. Cdk1p also contributes to Nup53p phosphorylation as specific inhibition of Cdk1p or mutation of Cdk1p consensus sites partially blocked its phosphorylation. The ability of nup53 alleles containing Cdk1p site mutations to complement synthetic defects of nup53 Delta nup170 Delta strains is linked to a function for Nup53p in the spindle assembly checkpoint.     

DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response.     

Structural constraints on autoprocessing of the human nucleoporin Nup98

Nucleoporin Nup98, a 98-kDa protein component of the nuclear pore complex, plays an important role in both protein and RNA transport. During its maturation process, Nup98 undergoes post-translational autoproteolysis, which is critical for targeting to the NPC. Here we present high-resolution crystal structures of the C-terminal autoproteolytic domains of Nup98 (2.3 A for the wild type and 1.9 A for the S864A precursor), and propose a detailed autoproteolysis mechanism through an N-O acyl shift. Structural constraints are found at the autocleavage site, and could thus provide a driving force for autocleavage at the scissile peptide bond. Such structural constraints appear to be generated, at least in part, by anchoring a conserved phenylalanine side chain into a highly conserved hydrophobic pocket at the catalytic site. Our high-resolution crystal structures also reveal that three highly conserved residues, Tyr866, Gly867, and Leu868, provide most of the interactions between the autoproteolytic domain and the C-terminal tail. These results suggest that Nup98 may represent a new subtype of protein that utilizes autoprocessing to control biogenesis pathways and intracellular translocation.     

Structural basis for the interaction between the Tap/NXF1 UBA domain and FG nucleoporins at 1A resolution

The mRNA nuclear export function of Tap/NXF1 requires interactions with nuclear pore proteins (nucleoporins) that contain characteristic Phe-Gly repeats based on FG, GLFG or FxFG cores separated by hydrophilic linkers. FG-nucleoporins bind the two most C-terminal domains of Tap, which have NTF2 and UBA folds, respectively. We used a combination of NMR and X-ray crystallography to define the interaction interface between Tap UBA and FxFG nucleoporins and show that it involves primarily the two aromatic rings of the FxFG core that bind in a hydrophobic surface depression centred on Tap Cys588. NMR evidence indicates that the same depression mediates the binding of GLFG nucleoporins, which we confirmed by demonstrating competition between the two classes of repeat for binding to Tap UBA. Moreover, modification of Cys588 reduced the binding of Tap UBA to both GLFG and FxFG nucleoporins as well as to nuclear envelopes. These data underscore the central role of the conserved FG-nucleoporin repeat cores in binding to Tap UBA and indicate that functional differences between different classes of nucleoporins depend more on their spatial distribution in nuclear pores than on their binding to different sites on Tap UBA.     

The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.     

Retrieval of Resident Late-Golgi Membrane Proteins from the Prevacuolar Compartment of Saccharomyces cerevisiae Is Dependent on the Function of Grd19p

The dynamic vesicle transport processes at the late-Golgi compartment of Saccharomyces cerevisiae (TGN) require dedicated mechanisms for correct localization of resident membrane proteins. In this study, we report the identification of a new gene, GRD19, involved in the localization of the model late-Golgi membrane protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and lumenal domains of the alkaline phosphatase [ALP]), which localizes to the yeast TGN. A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole. In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting. The recycling of the carboxypeptidase Y sorting receptor, Vps10p, between the TGN and the prevacuolar compartment is largely unaffected in grd19Δ cells. Kinetic assays of A-ALP trafficking indicate that GRD19 is involved in the process of retrieval of A-ALP from the prevacuolar compartment. GRD19 encodes a small hydrophilic protein with a predominantly cytosolic distribution. In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment. Using an in vitro binding assay, Grd19p was found to interact physically with the cytosolic domain of DPAP A. We conclude that Grd19p is a component of the retrieval machinery that functions by direct interaction with the cytosolic tails of certain TGN membrane proteins during the sorting/budding process at the prevacuolar compartment.     

Characterization of the antibodies to p62 nucleoporin in primary biliary cirrhosis using human recombinant antigen

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat-germ agglutinin (WGA)-Sepharose, was previously detected. Recently, clinical significance of the anti-NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti-NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti-p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N-acetylglucosamine residues. More than 50% of tested PBC sera precipitated (35)S-radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti-p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62-positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti-p62 in PBC.     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

The GLFG regions of Nup116p and Nup100p serve as binding sites for both Kap95p and Mex67p at the nuclear pore complex

Our previous studies have focused on a family of Saccharomyces cerevisiae nuclear pore complex (NPC) proteins that contain domains composed of repetitive tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We have previously shown that the GLFG regions of Nup116p and Nup100p directly bind the karyopherin transport factor Kap95p during nuclear protein import. In this report, we have further investigated potential roles for the GLFG region in mRNA export. The subcellular localizations of green fluorescent protein (GFP)-tagged mRNA transport factors were individually examined in yeast cells overexpressing the Nup116-GLFG region. The essential mRNA export factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus. In contrast, the localizations of Gle1-GFP and Gle2-GFP remained predominantly associated with the NPC, as in wild type cells. The localization of Kap95p was also not perturbed with GLFG overexpression. Coimmunoprecipitation experiments from yeast cell lysates resulted in the isolation of a Mex67p-Nup116p complex. Soluble binding assays with bacterially expressed recombinant proteins confirmed a direct interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions. Mtr2p was not required for in vitro binding of Mex67p to the GLFG region. To map the Nup116-GLFG subregion(s) required for Kap95p and/or Mex67p association, yeast two-hybrid analysis was used. Of the 33 Nup116-GLFG repeats that compose the domain, a central subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the full-length region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the capacity to bind both karyopherins and an mRNA export factor simultaneously. Taken together, our in vivo and in vitro results define an essential role for a direct Mex67p-GLFG interaction during mRNA export.     

Influence of cargo size on Ran and energy requirements for nuclear protein import

Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin alpha/beta and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin beta and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.     

The Nup2 meiotic-autonomous region relieves inhibition of Nup60 to promote progression of meiosis and sporulation in Saccharomyces cerevisiae

During meiosis, chromosomes undergo dramatic changes in structural organization, nuclear positioning, and motion. Although the nuclear pore complex has been shown to affect genome organization and function in vegetative cells, its role in meiotic chromosome dynamics has remained largely unexplored. Recent work in the budding yeast Saccharomyces cerevisiae demonstrated that the mobile nucleoporin Nup2 is required for normal progression through meiosis I prophase and sporulation in strains where telomere-led chromosome movement has been compromised. The meiotic-autonomous region, a short fragment of Nup2 responsible for its role in meiosis, was shown to localize to the nuclear envelope via Nup60 and to bind to meiotic chromosomes. To understand the relative contribution these 2 activities have on meiotic-autonomous region function, we first carried out a screen for meiotic-autonomous region mutants defective in sporulation and found that all the mutations disrupt interaction with both Nup60 and meiotic chromosomes. Moreover, nup60 mutants phenocopy nup2 mutants, exhibiting similar nuclear division kinetics, sporulation efficiencies, and genetic interactions with mutations that affect the telomere bouquet. Although full-length Nup60 requires Nup2 for function, removal of Nup60’s C-terminus allows Nup60 to bind meiotic chromosomes and promotes sporulation without Nup2. In contrast, binding of the meiotic-autonomous region to meiotic chromosomes is completely dependent on Nup60. Our findings uncover an inhibitory function for the Nup60 C-terminus and suggest that Nup60 mediates recruitment of meiotic chromosomes to the nuclear envelope, while Nup2 plays a secondary role counteracting the inhibitory function in Nup60’s C-terminus.     

A Role for the Karyopherin Kap123p in Microtubule Stability

Several components of the nuclear transport machinery play a role in mitotic spindle assembly in higher eukaryotes. To further investigate the role of this family of proteins in microtubule function, we screened for mutations in Saccharomyces cerevisiae that confer sensitivity to microtubule-destabilizing drugs. One mutant exhibiting this phenotype lacked the gene encoding the karyopherin Kap123p. Analysis of kap123 Δ cells revealed that the drug sensitivity was caused by a defect in microtubule stability and/or assembly. In support of this idea, we demonstrated genetic interactions between the kap123 Δ mutation and mutated alleles of genes encoding α-tubulins and factors controlling microtubule dynamics. Moreover, kap123 Δ cells exhibit defects in spindle structure and dynamics as well as nuclear positioning defects during mitosis. Cultures of kap123 Δ strains are enriched for mononucleated large-budded cells often containing short spindles and nuclei positioned away from the budneck, phenotypes indicative of defects in both cytoplasmic and nuclear microtubules. Finally, we identified a gene, CAJ1 , which when deleted in combination with KAP123 exacerbated the microtubule-related defects of the kap123 Δ mutants. We propose that Kap123p and Caj1p, a member of the Hsp40 family of proteins, together play an essential role in normal microtubule function.     

Nuclear import of the ran exchange factor, RCC1, is mediated by at least two distinct mechanisms

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an approximately 45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH(2)-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-alpha3 isoform of importin-alpha. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-alpha or importin-beta or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.