Genetic Analysis of Macromolecular Transport across the Nuclear Envelope (original) (raw)
Cell, 2004
identified, including components of the nuclear pore complex (NPC; Feuerbach et al., 2002). The S. cerevisiae NPC is 05ف MDa in size, comprised of 03ف nucleoporin proteins, and is the channel through which proteins traverse the nuclear envelope. The nuclear face of the NPC is arranged in a basket-like structure, extending 59ف nm of Cancer Biology into the nucleoplasm (Fahrenkrog et al., 1998). Several The Dana-Farber Cancer Institute studies have demonstrated that nucleoporins are essen-Boston, Massachusetts 02115 tial for mediating epigenetic control of transcription in yeast. Feuerbach et al. have shown that the nucleoporins NUP60 and NUP145 are genetically required for full Summary repression of the HMR locus. The deletion of myosinlike protein genes MLP1 and MLP2, which are nuclear The association of genes with the nuclear pore compore associated (Galy et al., 2000; Kosova et al., 2000; plex (NPC) and nuclear transport factors has been Strambio-de-Castillia et al., 1999), also causes dereimplicated in transcriptional regulation. We therefore pression of a reporter gene at the HMR locus (Feuerbach examined the association of components of the nuet al., 2002). Furthermore, MLP1 and MLP2 have been clear transport machinery including karyopherins, implicated in the control of telomere anchoring and renucleoporins, and the Ran guanine-nucleotide exchange pression (Galy et al., 2000; Hediger et al., 2002). A recent factor (RanGEF) with the Saccharomyces cerevisiae screen for boundary proteins that prevent the spread of genome. We find that most nucleoporins and karyosilencing at the HML locus identified several compopherins preferentially associate with a subset of highly nents of the nuclear transport machinery including the transcribed genes and with genes that possess Rap1 exporter, Cse1, and the nucleoporin Nup2 (Ishii et al., binding sites whereas the RanGEF preferentially asso-2002). Nup2 is a known docking site for Cse1 (Hood ciates with transcriptionally inactive genes. Consistent et al., 2000), and the absence of Nup2 from the NPC with coupling of transcription to the nuclear pore, we abolishes the boundary activity of Cse1, highlighting the show that transcriptional activation of the GAL genes influence of nuclear transport factors on transcriptional results in their association with nuclear pore proteins, state (Ishii et al., 2002). These results have suggested relocation to the nuclear periphery, and loss of that association of genes with the NPC may play a key RanGEF association. Taken together, these results inrole in transcriptional regulation, possibly by topologidicate that the organization of the genome is coupled cally constraining repressed DNA segments. via transcriptional state to the nuclear transport ma-The mammalian Ran guanine-nucleotide exchange chinery. factor, or RanGEF, is known to associate with a nuclear basket nucleoporin, Nup98, and with chromatin (Fon-Introduction toura et al., 2000; Nemergut et al., 2001). RanGEF mutants exhibit defects in nuclear structure, chromosome Transcriptional regulation has been shown to correlate stability, and chromatin condensation as well as perturwith the intranuclear position of genes in a variety of bation of nuclear transport (Aebi et al., 1990; Azuma and species. For example, proper silencing of genes in-Dasso, 2000; Clark et al., 1991; Forrester et al., 1992; volved in B cell and T cell development is dependent Kadowaki et al., 1994; Ohtsubo et al., 1987). As such, upon the ability of the genes to relocate to centromeric NPC components and the RanGEF are well-established heterochromatin (Brown et al., 1997, 1999). In Drosophcontributors to genomic organization in S. cerevisiae. ila melanogaster, the degree of position effect variega-Using genomic location analysis (Ren et al., 2000), we tion is correlated with the subnuclear localization of a have examined all genes bound by the NPC, several reporter gene (Dernburg et al., 1996). Additionally, karyopherins, and the RanGEF in S. cerevisiae. We find proper silencing of the mating-type loci in Saccharothat the silent mating-type loci, subtelomeric genes, and myces cerevisiae, HML and HMR, is dependent upon many transcriptionally active genes can be found in astheir ability to associate with the nuclear periphery (Ansociation with the NPC. Further analysis demonstrates drulis et al., 1998, 2002; Feuerbach et al., 2002). Furtherthat NPC-associated genes are significantly enriched more, the HML locus is derepressed when relocated for the binding site of the transcriptional regulator Rap1. from its subtelomeric site to a more centromere-proxi-Additionally, members of nuclear transport complexes mal location or to the arm of a different chromosome with similar functional roles have similar occupancy pro-(Maillet et al., 1996; Marcand et al., 1996; Stavenhagen files. Finally, we find both by chromatin immunoprecipiand Zakian, 1994; Thompson et al., 1994). Thus, the tation and microscopy that genes relocate from the nuspatial context of a gene within the nucleus as well cleoplasm to the nuclear pore upon transcriptional as within a chromosome appears to be critical in the induction. epigenetic control of heterochromatin formation. A number of nuclear proteins important for the mainte-Results nance of higher order genomic organization have been The nucleoporins Nup2, Nup60, and Nup145, the karyopherin Cse1, and the nuclear pore-associated proteins *Correspondence: pamela_silver@dfci.harvard.edu Cell 428 Consortium. Nat. Genet. 25, 25-29. Azuma, Y., and Dasso, M. (2000). The role of Ran in nuclear function. Curr. Opin. Cell Biol. 12, 302-307. Combined FISH/IF A 5.3 kb fragment spanning the coding sequence of GAL1, GAL7, Becskei, A., and Mattaj, I.W. (2003). The strategy for coupling the and GAL10 was amplified from genomic DNA using the following RanGTP gradient to nuclear protein export. Proc. Natl. Acad. Sci. primer sequences: 5Ј-CATTTGGGCCCCCTGGAACC-3Ј and 5Ј-USA 100, 1717-1722. GGGGCTAAAACATATGACGAAACA-3Ј. The digoxigenin-dUTP de-Berriz, G.F., King, O.D., Bryant, B., Sander, C., and Roth, F.P. (2003). rivatized GAL probe was resuspended in hybridization solution (50% Characterizing gene sets with FuncAssociate. Bioinformatics 19, formamide, 10% dextran sulfate, 2x SSC) to a final concentration 2502-2504. of 01ف ng/ml. Combined IF/FISH was performed using a modified Blobel, G. (1985). Gene gating: a hypothesis. Proc. Natl. Acad. Sci. protocol based on a previously described technique (Gotta et al., USA 82, 8527-8529. 1999). Cells were grown in rich media containing either 2% glucose Booth, J.W., Belanger, K.D., Sannella, M.I., and Davis, L.I. (1999). or 2% galactose at 30ЊC to a density of 1ف ϫ 10 7 cells/ml then fixed The yeast nucleoporin Nup2p is involved in nuclear export of imin 4% paraformaldehyde before spheroplasting to prevent nuclear portin alpha/Srp1p. J. Biol. Chem. 274, 32360-32367. spreading. The anti-nucleoporin antibody, MAb414 (Covance), was used at a 1:5000 dilution. Pre-absorbed Alexa Fluor 594 goat anti-Brown, K.E., Guest, S.S., Smale, S.T., Hahm, K., Merkenschlager, mouse (Molecular Probes) and sheep anti-digoxigenin-fluorescein M., and Fisher, A.G. (1997). Association of transcriptionally silent (Roche) were used at 1:50 dilutions. Cells were imaged using a genes with Ikaros complexes at centromeric heterochromatin. Cell Nikon TE2000U inverted microscope with PerkinElmer ultraview 91, 845-854. spinning disk confocal. Confocal z sections were encoded by the Brown, K.E., Baxter, J., Graf, D., Merkenschlager, M., and Fisher, authors before being blindly evaluated by others. A stringent require-A.G. (1999). Dynamic repositioning of genes in the nucleus of lymment for complete overlap with the NPC was used to score GAL phocytes preparing for cell division. Mol. Cell 3, 207-217. region localization at the nuclear periphery. Error bars represent Buchman, A.R., Kimmerly, W.J., Rine, J., and Kornberg, R.D. (1988). variation in scoring in multiple blind tests. Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating se-Acknowledgments quences, and telomeres in Saccharomyces cerevisiae. Mol. Cell. Biol. 8, 210-225. The authors would like to thank I. Simon for help in developing our Clark, K.L., Ohtsubo, M., Nishimoto, T., Goebl, M., and Sprague, genomic localization procedure; P. Grosu, R. Gali, and the Bauer G.F., Jr. (1991). The yeast SRM1 protein and human RCC1 protein Center for Genomics Research, Harvard University, for help with share analogous functions. Cell Regul. 2, 781-792. microarray analysis; Rosetta Biosoftware for help with the Rosetta Damelin, M., Simon, I., Moy, T.I., Wilson, B., Komili, S., Tempst, P., Resolver microarray analysis platform; bass, U. (2002). Nuclear architecture and spatial positioning help and Gasser, S.M. (1992). Localization of RAP1 and topoisomerase II establish transcriptional states of telomeres in yeast. Nat. Cell Biol. in nuclei and meiotic chromosomes of yeast. J. Cell Biol. 117, 4, 214-221. 935-948. Fontoura, B.M., Blobel, G., and Yaseen, N.R. (2000). The nucleoporin Knop, M., Siegers, K., Pereira, G., Zachariae, W., Winsor, B., Na-Nup98 is a site for GDP/GTP exchange on ran and termination of smyth, K., and Schiebel, E. (1999). Epitope tagging of yeast genes karyopherin beta 2-mediated nuclear import. J. Biol. Chem. 275, using a PCR-based strategy: more tags and improved practical 31289-31296. routines. Yeast 15, 963-972. Forrester, W., Stutz, F., Rosbash, M., and Wickens, M. (1992). De-Kosova, B., Pante, N., Rollenhagen, C., Podtelejnikov, A., Mann, M., fects in mRNA 3Ј-end formation, transcription initiation, and mRNA Aebi, U., and Hurt, E. (2000). Mlp2p, a component of nuclear pore transport associated with the yeast mutation prp20: possible couattached intranuclear filaments, associates with nic96p. J. Biol. pling of mRNA processing and chromatin structure. Genes...
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.
Studying nuclear protein import in yeast
Methods, 2006
The yeast Saccharomyces cerevisiae is a common model organism for biological discovery. It has become popularized primarily because it is biochemically and genetically amenable for many fundamental studies on eukaryotic cells. These features, as well as the development of a number of procedures and reagents for isolating protein complexes, and for following macromolecules in vivo, have also fueled studies on nucleo-cytoplasmic transport in yeast. One limitation of using yeast to study transport has been the absence of a reconstituted in vitro system that yields quantitative data. However, advances in microscopy and data analysis have recently enabled quantitative nuclear import studies, which, when coupled with the signiWcant advantages of yeast, promise to yield new fundamental insights into the mechanisms of nucleo-cytoplasmic transport.
Proceedings of The National Academy of Sciences, 1998
We sublocalized the yeast nucleoporin Nup82 to the cytoplasmic side of the nuclear pore complex (NPC) by immunoelectron microscopy. Moreover, by in vitro binding assays we showed that Nup82 interacts with the C-terminal region of Nup159, a yeast nucleoporin that previously was also localized to the cytoplasmic side of the NPC. Hence, the two nucleoporins, Nup82 and Nup159, form a cytoplasmically oriented subcomplex that is likely to be part of the fibers emanating from the cytoplasmic ring of the NPC. Overexpression of Rss1/Gle1, a putative nucleoporin and/or mRNA transport factor, was shown previously to partially rescue depletion of Nup159. We show here that overexpression of Rss1/Gle1 also partially rescued depletion of Nup82. Depletion of either Nup82, Nup159, or Rss1/Gle1 was shown previously to inhibit mRNA export. As was reported previously for depletion of Nup159 or of Rss1/Gle1, we show here that depletion of Nup82 has no detectable effect on classical nuclear localization sequence-mediated nuclear import. In summary, the nucleoporins Nup159 and Nup82 form a cytoplasmically oriented subcomplex of the NPC that is likely associated with Rss1/Gle1; this complex is essential for RNA export, but not for classical nuclear localization sequence-mediated nuclear protein import.
Nuclear export dynamics of RNA–protein complexes
Nature, 2011
The central dogma of molecular biology-DNA makes RNA makes proteins-is a flow of information that in eukaryotes encounters a physical barrier: the nuclear envelope, which encapsulates, organizes and protects the genome. Nuclear-pore complexes, embedded in the nuclear envelope, regulate the passage of molecules to and from the nucleus, including the poorly understood process of the export of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work. Since its first description in electron micrographs 1 , our understanding of the nuclear-pore complex (NPC), arguably the largest nanomachine in the cell, has increased steadily. We are now at the point where we have a comprehensive overview of the NPC components and their contribution to its structure, as well as initial insights into the mechanism of NPC assembly and a sound understanding of the principal functions of the NPC 2. The 100-nm diameter NPC has a core structure consisting of a hollow cylinder embedded in the nuclear envelope, which displays an eight-fold symmetry of about 30 different proteins termed nucleoporins (Nups). The NPC acts as the gateway between the nucleus and the cytoplasm; only those macromolecules carrying specific import and export signals are permitted to pass through the central channel of the NPC, although water and metabolites can pass through freely 3,4. The NPC consists of several major domains (Fig. 1): the selective central channel, or central transporter region; the core scaffold that supports the central channel; the transmembrane regions; the nuclear basket; and the cytoplasmic filaments 5. The central channel is filled and surrounded with a distinct class of Nup that has numerous large domains rich in phenylalanine and glycine repeats, termed FG Nups. It is this central channel and the FG Nups that seem sufficient to mediate selective receptor-mediated transport 6,7. The nuclear basket consists of eight filaments that reach into the nucleoplasm, attached to each other by a ring at the end. Electron microscopy tomographs have shown that filaments extend from this basket into the nucleus 8,9. The cytoplasmic filaments are less ordered, forming highly mobile molecular rods projecting into the cytoplasm. The reach of NPCs can extend about 100 nm into the nucleus and cytoplasm 10,11 .
Journal of Cell Biology, 2002
he nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cyto-T plasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin ␣ /  -or transportin-dependent import.