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An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.

NPCs are the sole sites of exchange between the nucleus and cytoplasm. A large family of transport factors carry cargo between the nucleus and cytoplasm through the NPC. The NPC is a huge symmetric octagonal structure comprised of dozens of NUPs. NUPs make many contacts with surrounding structures, including the NE, the cytoplasm and nuclear interior. A subset of NUPs contain repeated peptide motifs that serve as docking sites for transport factors. The directionality of transport is determined by the transport factor, and its interactions with the small GTPase Ran and NUPs. Very little is known about how the NPC mediates transport, NPC assembly and the NPC's role in regulating transport, but these areas of research are beginning to emerge.

The 26 kD basic subunit of 280 kD buckwheat grain legumin has been partially characterized by measurement of its fluorescence and CD spectra. The protein has 22% alpha-helix, 36% beta-sheet, 12% beta-turn and 30% random coil secondary structure. In comparison with the basic subunits of other legumin-type proteins, the buckwheat legumin subunit has a high content of lysine and methionine. The protein also has higher ratios of lysine to arginine and methionine to arginine.

While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC.

We report a novel connection between nuclear pore complexes (NPCs) and spindle pole bodies (SPBs) revealed by our studies of the Saccharomyces cerevisiae NDC1 gene. Although both NPCs and SPBs are embedded in the nuclear envelope (NE) in yeast, their known functions are quite distinct. Previous work demonstrated that NDC1 function is required for proper SPB duplication (Winey, M., M.A. Hoyt, C. Chan, L. Goetsch, D. Botstein, and B. Byers. 1993. J. Cell Biol. 122:743-751). Here, we show that Ndc1p is a membrane protein of the NE that localizes to both NPCs and SPBs. Indirect immunofluorescence microscopy shows that Ndc1p displays punctate, nuclear peripheral localization that colocalizes with a known NPC component, Nup49p. Additionally, distinct spots of Ndc1p localization colocalize with a known SPB component, Spc42p. Immunoelectron microscopy shows that Ndc1p localizes to the regions of NPCs and SPBs that interact with the NE. The NPCs in ndc1-1 mutant cells appear to function normally at the nonpermissive temperature. Finally, we have found that a deletion of POM152, which encodes an abundant but nonessential nucleoporin, suppresses the SPB duplication defect associated with a mutation in the NDC1 gene. We show that Ndc1p is a shared component of NPCs and SPBs and propose a shared function in the assembly of these organelles into the NE.

In eukaryotic cells, a regulated flux of molecules between the cytoplasm and the nucleus maintains two very different environments while allowing the controlled exchange of macromolecules necessary for their individual functions. Molecules entering or leaving the nucleus use nuclear localization signals or nuclear export signals to pass through selective channels in the nuclear envelope formed by nuclear pore complexes. The recognition of signal-bearing cargo, its interaction with the nuclear pore complex and its translocation through the pore complex are mediated by soluble transport factors. Recently, the list of potential transport factors has grown rapidly, suggesting a previously unanticipated level of complexity for nuclear transport.

We have calculated a three-dimensional map of the yeast nuclear pore complex (yNPC) from frozen-hydrated specimens, thereby providing a direct comparison with the vertebrate NPC. Overall, the smaller yNPC is comprised of an octagonal inner spoke ring that is anchored within the nuclear envelope by a novel membrane-interacting ring. In addition, a cylindrical transporter is located centrally within the spokes and exhibits a variable radial expansion in projection that may reflect gating. The inner spoke ring, a transmembrane spoke domain, and the transporter are conserved between yeast and vertebrates; hence, they are required to form a functional NPC. However, significant alterations in NPC architecture have arisen during evolution that may be correlated with differences in nuclear transport regulation or mitotic behavior.

The spindle pole body (SPB) is the microtubule organizing center (MTOC) in the yeast Saccharomyces that plays a pivotal role in such diverse processes as mitosis, budding, and mating. We have used cryoelectron microscopy and image processing to study the structure of isolated diploid SPBs. We show that SPBs are present in two lateral-size classes, sharing a similar vertical architecture comprised of six major layers. Tomographic reconstructions of heparin-stripped SPBs reveal a central hexagonally packed layer. Overexpression of Spc42p results in the growth of a similar layer, forming a crystal that encircles the SPB. Hence, the SPB is an MTOC that utilizes crystallographic packing of subunits in its construction.

Protein transport into the nucleus is governed by the interaction of soluble transport factors with their import substrates and nuclear pore complexes. Here, we identify a major distinct nuclear import pathway, mediated by a previously uncharacterized yeast beta karyopherin Kap123p. The predominant substrates for this pathway are ribosomal proteins, which must be imported into the nucleus prior to assembly into pre-ribosomes. Kap123p binds directly to its transport substrates, repeat motif-containing nucleoporins, and Ran-GTP. We show that the related protein Pse1p is also a karyopherin and can functionally substitute for Kap123p; both are capable of specifically directing a ribosomal nuclear localization signal reporter to the nucleus in vivo.

A cytosolic yeast karyopherin, Kap104p, was isolated and shown to function in the nuclear import of a specific class of proteins. The protein bound directly to repeat-containing nucleoporins and to a cytosolic pool of two nuclear messenger RNA (mRNA) binding proteins, Nab2p and Nab4p. Depletion of Kap104p resulted in a rapid shift of Nab2p from the nucleus to the cytoplasm without affecting the localization of other nuclear proteins tested. This finding suggests that the major function of Kap104p lies in returning mRNA binding proteins to the nucleus after mRNA export.

A high molecular weight (HMW) globulin, the main storage protein in grains of the common buckwheat (Fagopyrum esculentum Moench) has been partially characterized. The protein exists as an oligomeric complex with molecular mass of 280 kD and is composed of three groups (alpha, beta and gamma of polypeptides with molecular weights ranging between 55-60, 32-44 and 16-29 kD respectively. The protein showed serological cross reactivity with globulins of soya bean, mung bean and pea. Antibodies raised against the purified protein did not recognize the storage proteins from broad bean, rice, wheat and grain amaranth. The 280 kD globulin of buckwheat grains belongs to the legumin family of proteins.

We have isolated a major protein constituent from a highly enriched fraction of yeast nuclear pore complexes (NPCs). The gene encoding this protein, Nup188p, was cloned, sequenced, and found to be nonessential upon deletion. Nup188p cofractionates with yeast NPCs and gives an immunofluorescent staining pattern typical of nucleoporins. Using immunoelectron microscopy, Nup188p was shown to localize to both the cytoplasmic and nucleoplasmic faces of the NPC core. There, Nup188p interacts with an integral protein of the pore membrane domain, Pom152p, and another abundant nucleoporin, Nic96p. The effects of various mutations in the NUP188 gene on the structure of the nuclear envelope and the function of the NPC were examined. While null mutants of NUP188 appear normal, other mutants allelic to NUP188 exhibit a dominant effect leading to the formation of NPC-associated nuclear envelope herniations and growth inhibition at 37 degrees C. In addition, depletion of the interacting protein Pom152p in cells lacking Nup188p resulted in severe deformations of the nuclear envelope. We suggest that Nup188p is one of a group of proteins that form the octagonal core structure of the NPC and thus functions in the structural organization of the NPC and nuclear envelope.

To extend our understanding of the mechanism by which the nuclear pore complex (NPC) mediates macromolecular transport across the nuclear envelope we have focused on defining the composition and molecular organization of the yeast NPC. Peptide sequence analysis of a polypeptide with a M(r) of approximately 100,000 present in a highly enriched yeast NPC fraction identified a novel yeast nucleoporin we term Nup120p. Nup120p corresponds to the open reading frame (ORF) YKL057c identified by the yeast genome sequencing project. The ORF predicts a protein with a calculated molecular mass of 120.5 kD containing two leucine zipper motifs, a short coiled-coil region and limited primary sequence similarity to Nup133p. Nup120p was localized to the NPC using a protein A-tagged chimera in situ by indirect immunofluorescence microscopy. Deletion of the NUP120 gene caused clustering of NPCs at one side of the nuclear envelope, moderate nucleolar fragmentation and slower cell growth. Transfer of nup120 delta cells to 37 degrees C resulted in the nuclear accumulation of poly(A)+ mRNA, extensive fragmentation of the nucleolus, spindle defects, and cell death.

We have taken a combined genetic and biochemical approach to identify major constituents of the yeast nuclear pore complex (NPC). A synthetic lethal screen was used to identify proteins which interact genetically with the major pore-membrane protein Pom152p. In parallel, polypeptides present in similar amounts to Pom152p in a highly enriched preparation of yeast NPCs have been characterized by direct microsequencing. These approaches have led to the identification of two novel and major nucleoporins, Nup170p and Nup157p. Both Nup170p and Nup157p are similar to each other and to an abundant mammalian nucleoporin, Nup155p (Radu, A., G. Blobel, and R. W. Wozniak. 1993. J. Cell Biol. 121: 1-9) and interestingly, nup170 mutants can be complemented with mammalian NUP155. In addition, the synthetic lethal screen identified genetic interactions between Pom152p and two other major nucleoporins, Nup188p (Nehrbass, U., S. Maguire, M. Rout, G. Blobel, and R. W. Wozniak, manuscript submitted for publication), and Nic96p (Grandi, P., V. Doye, and E. C. Hurt. 1993. EMBO J. 12: 3061-71). We have determined that together, Nup170p, Nup157p, Pom152p, Nup188p, and Nic96p comprise greater than one-fifth of the mass of the isolated yeast NPC. Examination of the genetic interactions between these proteins indicate that while deletion of either POM152, NUP170, or NUP188 alone is not lethal, pairwise combinations are. Deletion of NUP157 is also not lethal. However, nup157 null mutants, while lethal in combination with nup170 and nup188 null alleles, are not synthetically lethal with pom152 null alleles. We suggest that Nup170p and Nup157p may be part of a morphologically symmetrical but functionally distinct substructure of the yeast NPC, e.g., the nucleoplasmic and cytoplasmic rings. Finally, we observed morphological abnormalities in the nuclear envelope as a function of alterations in the expression levels of NUP170 suggesting a specific stoichiometric relationship between NPC components is required for the maintenance of normal nuclear structure.

We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems.

We have identified a new yeast nucleoporin of 159 kDa that we term NUP159. Immunofluorescence microscopy with a monospecific monoclonal antibody against NUP159 gave the punctate nuclear rim staining characteristic of nucleoporins. Immunogold electron microscopy with isolated yeast NEs yielded decoration of only the cytoplasmic side of the nuclear pore complex. The gene encoding NUP159 is essential, and, like some other nucleoporins, NUP159 contains a coiled-coil domain as well as a domain of repeated motifs. Five segments of NUP159, covering its entire length, were expressed in Escherichia coli. The repeat motif-containing segment was found to bind a nuclear transport substrate in the presence of vertebrate cytosolic extract containing nuclear transport factors. This segment also bound 35S-labeled mammalian karyopherin beta, one such transport factor that mediates the docking of substrates to the nuclear pore complex. These data establish a direct biochemical link between the repeat motif domain of a yeast nucleoporin, transport factors, (specifically karyopherin beta), and nuclear transport substrates. Its cytoplasmic aspect implies a role for NUP159 in nuclear import.

We have characterized a protein with an estimated molecular mass of 130 kDa that is contained in a highly enriched yeast nuclear pore complex (NPC) fraction. Partial amino acid sequence from this protein has led us to a previously identified open reading frame on chromosome XI of Saccharomyces cerevisiae encoding a protein of 133 kDa. Due to its coenrichment with NPCs during cell fractionation and the phenotype observed in the disrupted strain, we propose to term the gene encoding this protein NUP133. Cells carrying a disrupted copy of NUP133 were temperature sensitive for growth. In addition, abnormal clustering of NPCs was observed. This phenotype is similar to that previously observed in the disruption of another nucleoporin gene, NUP145. We speculate that the gene product of NUP133, Nup133p, may functionally overlap with the NUP145 gene product, Nup145p, and that these proteins may be involved in maintaining the position of the NPC within the nuclear envelope.

Nuclear pore complexes (NPCs) are enormous macromolecular structures that mediate the active exchange of proteins and RNPs between the nucleus and cytoplasm. Recent work has resulted in a windfall of identified NPC polypeptides, many with unique sequences. Several of the proteins have been shown to be part of extended cytoplasmic and nucleoplasmic NPC filaments. Biochemical, structural and genetic studies on NPC proteins are just beginning to allow an understanding of how they associate into a functional organelle.

We have identified a concanavalin A-reactive glycoprotein of 150 kD that coenriches with isolated yeast nuclear pore complexes. Molecular cloning and sequencing of this protein revealed a single canonical transmembrane segment. Epitope tagging and localization by both immunofluorescence and immunoelectron microscopy confirmed that it is a pore membrane protein. The protein was termed POM152 (for pore membrane protein of 152 kD) on the basis of its location and cDNA-deduced molecular mass. POM152 is likely to be a type II membrane protein with its NH2-terminal region (175 residues) and its COOH-terminal region (1,142 residues) positioned on the pore side and cisternal side of the pore membrane, respectively. The proposed cisternally exposed domain contains eight repetitive motifs of approximately 24 residues. Surprisingly, POM152 deletion mutants were viable and their growth rate was indistinguishable from that of wild-type cells at temperatures between 17 and 37 degrees C. However, overproduction of POM152 inhibited cell growth. When expressed in mouse 3T3 cells, POM152 was found to be localized to the pore membrane, suggesting a conserved sorting pathway between yeast and mammals.

Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of approximately 310 S and an M(r) of approximately 66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins.

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.

Yeast spindle pole bodies (SPBs) with attached nuclear microtubles were enriched approximately 600-fold from yeast cell extracts. 14 mAbs prepared against this enriched SPB fraction define at least three components of the SPB and spindle. Immunofluorescent staining of yeast cells showed that throughout the cell cycle two of the components (110 and 90 kD) were localized exclusively to the SPB region, and the other (80 kD) was localized both to the SPB region and to particulate dots in short spindles. Immunoelectron microscopy confirmed and extended most of these findings. Thus the 110-kD component was localized to a layer in the SPB just to the nuclear side of the plane of the inner nuclear membrane. The 90-kD component was localized in a layer across the cytoplasmic face of intact SPBs, and, in SPBs where nuclear microtubules were removed by extraction with DEAE-dextran, the 90-kD component was also found in an inner nuclear layer close to where spindle microtubules emerge. In intact SPBs with attached nuclear microtubules the anit-80-kD mAb labels microtubules, particularly those close to the SPB. These results begin to provide a preliminary molecular map of the SPB and should also enable the corresponding genes to be isolated.

In a three-year prospective study of acute cerebrovascular accident patients admitted to a geriatric unit within 72 hours of the onset, 12.7% had what was considered to be an associated acute myocardial infarction. In the majority of cases, 71%, there was no clinical indication of an acute myocardial infarction and, had it not been for electrocardiographic and enzyme studies, the concurrence of these two conditions might not have come to light. The possible explanations for the concurrence of these two conditions are explored. Patients were followed-up for five years. The mortality rate for the combined acute myocardial and cerebral infarction cases admitted to hospital was 53% in six weeks, and 64% in one year, compared with 26% and 42%, respectively, in those strokes uncomplicated by a cardiac infarction. Only one patient has survived for five years. The recognition of such cases is important in terms of management and prognosis and this may be achieved by ECG recordings followed by enzyme studies in all cases of acute stroke admitted to hospital.

Lumbar cerebrospinal fluid and arterial blood acid-base state were assessed in 19 patients within 24 hours of an acute cerebrovascular accident. Those with haemorrhage into the C.S.F. showed a lower C.S.F. pH and higher C.S.F. lactate than those without haemorrhage but the Pco(2), was similar in the two groups, suggesting that this greater C.S.F. acidity was not responsible for a greater degree of hyperventilation. In those without haemorrhage an inverse relation was found between C.S.F. pH and arterial Pco(2), suggesting that a non-chemical ventilatory drive-for example, due to central neurological damage-was responsible for the acid-base changes observed.

Respiratory pattern and arterial blood gas tensions were assessed in patients with acute cerebrovascular accidents. Hyperventilation, low Pco(2), and high arterial pH were associated with a poor prognosis, whereas patients with normal respiratory pattern and blood gas tensions survived. Periodic and Cheyne-Stokes breathing carried an intermediate prognosis.