12 results on '"Rout, M."'
Search Results
2. The yeast nuclear pore complex: composition, architecture, and transport mechanism.
- Author
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Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, and Chait BT
- Subjects
- Biological Transport, Carrier Proteins analysis, Carrier Proteins chemistry, Carrier Proteins genetics, Chromatography, High Pressure Liquid, Fluorescent Antibody Technique, Fungal Proteins chemistry, Fungal Proteins genetics, Membrane Proteins chemistry, Membrane Proteins genetics, Microscopy, Immunoelectron, Models, Biological, Molecular Weight, Nuclear Envelope genetics, Nuclear Envelope ultrastructure, Nuclear Proteins chemistry, Nuclear Proteins genetics, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Fungal Proteins analysis, Membrane Proteins analysis, Nuclear Envelope chemistry, Nuclear Envelope metabolism, Nuclear Proteins analysis, Saccharomyces cerevisiae ultrastructure
- Abstract
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.
- Published
- 2000
- Full Text
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3. Proteins connecting the nuclear pore complex with the nuclear interior.
- Author
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Strambio-de-Castillia C, Blobel G, and Rout MP
- Subjects
- Amino Acid Sequence, Biological Transport, Cloning, Molecular, Fungal Proteins chemistry, Fungal Proteins genetics, Gene Deletion, Green Fluorescent Proteins, Luminescent Proteins metabolism, Microscopy, Electron, Molecular Sequence Data, Nuclear Pore Complex Proteins, Nuclear Proteins chemistry, Nuclear Proteins genetics, RNA-Binding Proteins, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae metabolism, Sequence Homology, Amino Acid, Cell Nucleus metabolism, Fungal Proteins metabolism, Nuclear Envelope metabolism, Nuclear Proteins metabolism, Saccharomyces cerevisiae Proteins
- Abstract
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.
- Published
- 1999
- Full Text
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4. Saccharomyces cerevisiae Ndc1p is a shared component of nuclear pore complexes and spindle pole bodies.
- Author
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Chial HJ, Rout MP, Giddings TH, and Winey M
- Subjects
- Centrosome metabolism, Fluorescent Antibody Technique, Indirect, Gene Deletion, Macromolecular Substances, Membrane Glycoproteins deficiency, Membrane Glycoproteins genetics, Membrane Glycoproteins physiology, Microscopy, Immunoelectron, Nuclear Envelope metabolism, Nuclear Pore, Nuclear Pore Complex Proteins, Nuclear Proteins deficiency, Nuclear Proteins genetics, Nuclear Proteins physiology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Centrosome ultrastructure, Fungal Proteins metabolism, Membrane Proteins metabolism, Nuclear Envelope ultrastructure, Nuclear Proteins metabolism, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins
- Abstract
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.
- Published
- 1998
- Full Text
- View/download PDF
5. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications.
- Author
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Yang Q, Rout MP, and Akey CW
- Subjects
- Animals, Freezing, Fungal Proteins chemistry, Fungal Proteins ultrastructure, Image Processing, Computer-Assisted, Microscopy, Electron, Scanning Transmission, Molecular Weight, Protein Conformation, Saccharomyces metabolism, Species Specificity, Vertebrates, Evolution, Molecular, Nuclear Envelope chemistry, Nuclear Envelope ultrastructure, Saccharomyces chemistry, Saccharomyces ultrastructure
- Abstract
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.
- Published
- 1998
- Full Text
- View/download PDF
6. The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex.
- Author
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Nehrbass U, Rout MP, Maguire S, Blobel G, and Wozniak RW
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Genes, Fungal genetics, Genes, Lethal, Genetic Complementation Test, Membrane Glycoproteins analysis, Molecular Sequence Data, Molecular Weight, Mutation, Nuclear Envelope ultrastructure, Nuclear Pore, Nuclear Proteins chemistry, Nuclear Proteins isolation & purification, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins isolation & purification, Sequence Analysis, Sequence Analysis, DNA, Yeasts cytology, Yeasts genetics, Nuclear Envelope chemistry, Nuclear Pore Complex Proteins, Nuclear Proteins analysis, Nuclear Proteins genetics, Saccharomyces cerevisiae Proteins
- Abstract
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.
- Published
- 1996
- Full Text
- View/download PDF
7. Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport.
- Author
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Aitchison JD, Blobel G, and Rout MP
- Subjects
- Amino Acid Sequence, Base Sequence, Biological Transport genetics, Biomarkers, Fluorescent Antibody Technique, Gene Deletion, Genes, Fungal physiology, Microscopy, Electron, Molecular Sequence Data, Nuclear Envelope metabolism, Nuclear Envelope ultrastructure, Nuclear Proteins analysis, Nuclear Proteins genetics, RNA, Messenger metabolism, Sensitivity and Specificity, Temperature, Yeasts cytology, Yeasts ultrastructure, Nuclear Envelope chemistry, Nuclear Pore Complex Proteins, Nuclear Proteins isolation & purification, Saccharomyces cerevisiae Proteins, Yeasts genetics
- Abstract
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.
- Published
- 1995
- Full Text
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8. Isolation and characterization of nuclear envelopes from the yeast Saccharomyces.
- Author
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Strambio-de-Castillia C, Blobel G, and Rout MP
- Subjects
- Antibodies, Monoclonal, Biological Transport, Blotting, Western, Cell-Free System metabolism, Detergents pharmacology, Electrophoresis, Polyacrylamide Gel, Heparin pharmacology, Membrane Proteins metabolism, Microscopy, Electron, Nuclear Envelope immunology, Nuclear Envelope metabolism, Cell Fractionation methods, Nuclear Envelope ultrastructure, Saccharomyces cerevisiae ultrastructure
- Abstract
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.
- Published
- 1995
- Full Text
- View/download PDF
9. The essential yeast nucleoporin NUP159 is located on the cytoplasmic side of the nuclear pore complex and serves in karyopherin-mediated binding of transport substrate.
- Author
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Kraemer DM, Strambio-de-Castillia C, Blobel G, and Rout MP
- Subjects
- Amino Acid Sequence, Biological Transport, Molecular Sequence Data, Nuclear Proteins physiology, Cytoplasm chemistry, Fungal Proteins analysis, Nuclear Envelope chemistry, Nuclear Proteins analysis, Saccharomyces cerevisiae chemistry
- Abstract
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.
- Published
- 1995
- Full Text
- View/download PDF
10. Disruption of the nucleoporin gene NUP133 results in clustering of nuclear pore complexes.
- Author
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Pemberton LF, Rout MP, and Blobel G
- Subjects
- Fluorescent Antibody Technique, Membrane Proteins metabolism, Microscopy, Electron, Nuclear Envelope ultrastructure, Nuclear Proteins metabolism, Saccharomyces cerevisiae genetics, Membrane Proteins genetics, Nuclear Envelope metabolism, Nuclear Pore Complex Proteins, Nuclear Proteins genetics, Saccharomyces cerevisiae Proteins
- Abstract
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.
- Published
- 1995
- Full Text
- View/download PDF
11. POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope.
- Author
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Wozniak RW, Blobel G, and Rout MP
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Base Sequence, Fungal Proteins genetics, Genes, Fungal, Membrane Glycoproteins genetics, Mice, Molecular Sequence Data, Mutagenesis, Insertional, Nuclear Pore, Nuclear Proteins genetics, Repetitive Sequences, Nucleic Acid, Saccharomyces cerevisiae, Sequence Alignment, Sequence Homology, Amino Acid, Fungal Proteins metabolism, Membrane Glycoproteins metabolism, Nuclear Envelope chemistry, Nuclear Proteins metabolism, Saccharomyces cerevisiae Proteins
- Abstract
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.
- Published
- 1994
- Full Text
- View/download PDF
12. Isolation of the yeast nuclear pore complex.
- Author
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Rout MP and Blobel G
- Subjects
- Cell Fractionation, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Fungal Proteins isolation & purification, Microscopy, Electron, Nuclear Envelope chemistry, Nuclear Envelope ultrastructure, Saccharomyces cerevisiae ultrastructure
- Abstract
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.
- Published
- 1993
- Full Text
- View/download PDF
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