Evolutionary specialization of the nuclear targeting apparatus

Molecular and cellular biology, 1998

Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization ...

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: [email protected] 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...

Molecular and Cellular Biology, 2004

In yeast there are at least 14 members of the ␤-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions. Here, we have focused on the identification of nuclear import cargoes for the essential yeast ␤-karyopherin, Kap121p. Using an overlay blot assay and coimmunopurification studies, we have identified ϳ ϳ30 putative Kap121p cargoes. Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis. Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs. Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function. Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p. Together, our data elucidate additional levels of complexity in these nuclear transport pathways.

Cell, 1997

Recently, one such pathway was discovered. The karyopherin ␤ homolog Kap104p/transportin Howard Hughes Medical Institute The Rockefeller University imports a particular set of mRNA-binding proteins. Although it also docks to repeat motif-containing nucleo-New York, New York 10021 porins, it is unlike karyopherin ␤ in that it binds directly to its substrate (Aitchison et al., 1996; Pollard et al., 1996; Bonifaci et al., 1997). Summary Here, we demonstrate that Kap123p is a Saccharomyces karyopherin ␤, mediating a major alternative Protein transport into the nucleus is governed by the transport pathway. interaction of soluble transport factors with their import substrates and nuclear pore complexes. Here, we identify a major distinct nuclear import pathway, Results mediated by a previously uncharacterized yeast ␤ karyopherin Kap123p. The predominant substrates for Kap123p Is a ␤ Karyopherin Homolog this pathway are ribosomal proteins, which must be A detailed analysis of the composition of a highly enimported into the nucleus prior to assembly into preriched NPC fraction from the yeast Saccharomyces has ribosomes. Kap123p binds directly to its transport led to the identification of not only numerous nucleosubstrates, repeat motif-containing nucleoporins, and porins, but also transport factors presumably caught Ran-GTP. We show that the related protein Pse1p is during the NPC-associated phase of their cycle. Two also a karyopherin and can functionally substitute for known karyopherins were identified: Srp1p/Kap60p and Kap123p; both are capable of specifically directing a Kap95p (Yano et al., 1992; Enenkel et al., 1995). ribosomal nuclear localization signal reporter to the Sequence data from another protein in the fraction nucleus in vivo. identified an ORF in the genome database (YER110c), predicted to encode a polypeptide of 123 kDa. Because this protein proved functionally homologous to Kap95p

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.

Trends in Cell Biology, 2004

The importin a/b heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin a binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin b, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin a back to the cytoplasm. In this article, we discuss control mechanisms that importin a exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin a genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin a that seem to be distinct from its housekeeping functions in nuclear transport.

Cell cycle, 2004

The presence of a distinct nucleus, the compartment for confining the genome, transcription and RNA maturation, is a central (and eponymous) feature that distinguishes eukaryotes from prokaryotes. Structural integrity of the nucleus is maintained by the nuclear envelope (NE). A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication. We investigated the provenance of the conserved domains found in these perinuclear proteins and reconstructed a parsimonious scenario for NE and NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes. We show that the NE and NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, and de novo. It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1, and, probably, also the key domains of karyopherins and nucleoporins, the HEAT/ARM and WD40 repeats, have a bacterial, most likely, endosymbiotic origin. The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, and the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains. This suggests that independent, late horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages. Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mRNA-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions. It is shown that several NPC proteins containing super-structureforming α-helical and β-propeller modules are most closely related to corresponding proteins in the cytoplasmic vesicle biogenesis and coating complexes. From these observations, we infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the "prekaryote") as part of cell compartmentalization triggered by archaeo-bacterial symbiosis. A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding Ran, the key regulator of nuclear transport. A primitive NPC with approximately 20 proteins and a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred to have been integral perinuclear components in the las common ancestor of modern eukaryotes.

Current Opinion in Structural Biology, 2001

Molecular Genetics and Genomics, 2004

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the a-importin Srp1p and the b-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.

The Journal of Cell Biology, 1992

We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z. mays that specifically bind nuclear localization sequences (NLSs). These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S. cerevisiae. All NBPs are associated with nuclei and also present in the cytosol . NBPs are phosphorylated and phosphatase treatment abolished NLS binding . The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells . Proper import of T HE composition of the nucleus requires vectorial transport of macromolecules across the nuclear envelope. Thus far, import of proteins into the nucleus could be divided into several steps . The initial reaction includes specific recognition and binding of nuclear proteins at the nuclear envelope . This is followed by ATP-and temperaturedependent translocation through the nuclear pore complex .