Nup2p Dynamically Associates with the Distal Regions of the Yeast Nuclear Pore Complex (original) (raw)
Related papers
2001
The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleocytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin a/b-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating speci®c nuclear import events.
The Yeast Nuclear Pore Complex and Transport Through It
Genetics, 2012
Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell's genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or "Nups"), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC's role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins.
The Yeast Nuclear Pore Complex: Composition, Architecture, and Transport Mechanism
The Journal of Cell Biology, 2000
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.
Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport
The Journal of Cell Biology, 1995
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 nu...
The Journal of Cell Biology, 1996
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 Pom...
The Mechanism of Nucleocytoplasmic Transport through the Nuclear Pore Complex
Cold Spring Harbor Symposia on Quantitative Biology, 2010
Unlike their prokaryotic ancestors, eukaryotic cells contain numerous membrane-bound organelles that compartmentalize various specialized processes. This evolutionary advancement enabled tighter regulation of cellular functions but meant that cells needed to develop channels to transport proteins, ions, and other substances specifically across their internal and external membranes. Of all of the transport channels in a cell, one stands out from all of the others in terms of the range and size of cargoes that pass through it. This is the NPC, which mediates all transport across the nuclear envelope (NE) (the double-membraned layer that separates the nuclear genetic material from the cytoplasm). Transport through the NPC is rapid and bidirectional, with a large set of diverse cargoes crossing between the nucleus and cytoplasm. Cargoes such as transcription factors, ribosomal proteins, and viral nucleic acids enter the nucleus, whereas, messenger ribonucleoproteins (mRNPs), transfer RNAs (tRNAs), and ribosomal subunits exit. Despite the wide range of molecules that must pass through the NPC, nucleocytoplasmic transport is incredibly selective. Transport can also occur very quickly, up to the order of thousands of events per NPC per second (Ribbeck and Gorlich 2001). Yet the structure of the NPC, discussed below, has been found to be surprisingly simple. How this simple structure might mediate this rapid yet selective transport of such a variety of cargoes remains the main question in the field and is the major topic of this chapter. SOLUBLE PHASE OF TRANSPORT Karyopherin-Mediated Transport Numerous lines of evidence have shown that only proteins that can specifically bind to the family of nuclear pore proteins called FG nups are able to efficiently and rapidly cross the NPC. Many such FG-binding proteins act as transport factors, carrying non-FG-binding cargoes across the NPC. Karyopherins (Kaps), also known as importins and exportins, are a family of transport factors that carry proteins and protein/RNA complexes as large as 2 MDa across the NPC (Dworetzky and Feldherr 1988;
Molecular Architecture of the Yeast Nuclear Pore Complex: Localization of Nsp1p Subcomplexes
The Journal of Cell Biology, 1998
The nuclear pore complex (NPC), a supramolecular assembly of ف 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the threedimensional (3D) architecture of Xenopus NPCs, and eight of the ف 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.
2000
The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of ϳ30 different proteins (nucleoporins). Preembedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.
Journal of Biological Chemistry, 2004
Protein transport between the nucleus and cytoplasm requires interactions between nuclear pore complex proteins (nucleoporins) and soluble nuclear transport factors (karyopherins, importins, and exportins). Exactly how these interactions contribute to the nucleocytoplasmic transport of substrates remains unclear. Using a synthetic lethal screen with the nucleoporin NUP1, we have identified a conditional allele of NUP82, encoding an essential nuclear pore complex protein in Saccharomyces cerevisiae. This nup82-3 allele also exhibits synthetic genetic interactions with mutants of the karyopherin MSN5. nup82-3 mutants accumulate the Msn5 export substrate Pho4 within the nucleus at nonpermissive temperatures. The nuclear import of the RPA complex subunit Rfa2 is impaired in nup82-3 and in mutants of the karyopherin KAP95, but is not affected by the loss of MSN5. Interestingly, deletion of MSN5 results in retention of Rfa2-GFP within the nucleus under conditions in which it normally diffuses out. These data provide evidence that Nup82 is important for Msn5mediated nuclear protein export and Kap95-mediated protein import. In addition, Msn5 may play a role independent of import in the localization of Rfa2.