PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeEditorsContactInstructions for Authors
 
J Cell Biol. 1995 December 2; 131(6): 1677–1697.
PMCID: PMC2120651

Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene

Abstract

To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C. We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7- 1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p.

Full Text

The Full Text of this article is available as a PDF (6.0M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Akey CW. Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy. J Cell Biol. 1989 Sep;109(3):955–970. [PMC free article] [PubMed]
  • Akey CW. Visualization of transport-related configurations of the nuclear pore transporter. Biophys J. 1990 Aug;58(2):341–355. [PubMed]
  • Akey CW. Probing the structure and function of the nuclear pore complex. Semin Cell Biol. 1991 Jun;2(3):167–177. [PubMed]
  • Akey CW, Radermacher M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J Cell Biol. 1993 Jul;122(1):1–19. [PMC free article] [PubMed]
  • Allen JL, Douglas MG. Organization of the nuclear pore complex in Saccharomyces cerevisiae. J Ultrastruct Mol Struct Res. 1989 Aug;102(2):95–108. [PubMed]
  • Amberg DC, Goldstein AL, Cole CN. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Dev. 1992 Jul;6(7):1173–1189. [PubMed]
  • Amberg DC, Fleischmann M, Stagljar I, Cole CN, Aebi M. Nuclear PRP20 protein is required for mRNA export. EMBO J. 1993 Jan;12(1):233–241. [PubMed]
  • Aris JP, Blobel G. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J Cell Biol. 1988 Jul;107(1):17–31. [PMC free article] [PubMed]
  • Aris JP, Blobel G. Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins. J Cell Biol. 1989 Jun;108(6):2059–2067. [PMC free article] [PubMed]
  • Bassel J, Douglas HC. Osmotic remedial response in a galactose-negative mutant of Saccharomyces cerevisiae. J Bacteriol. 1968 Mar;95(3):1103–1110. [PMC free article] [PubMed]
  • Belanger KD, Kenna MA, Wei S, Davis LI. Genetic and physical interactions between Srp1p and nuclear pore complex proteins Nup1p and Nup2p. J Cell Biol. 1994 Aug;126(3):619–630. [PMC free article] [PubMed]
  • Bender A, Pringle JR. Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Mar;11(3):1295–1305. [PMC free article] [PubMed]
  • Bogerd AM, Hoffman JA, Amberg DC, Fink GR, Davis LI. nup1 mutants exhibit pleiotropic defects in nuclear pore complex function. J Cell Biol. 1994 Oct;127(2):319–332. [PMC free article] [PubMed]
  • Brandriss MC. Evidence for positive regulation of the proline utilization pathway in Saccharomyces cerevisiae. Genetics. 1987 Nov;117(3):429–435. [PubMed]
  • Brewster JL, de Valoir T, Dwyer ND, Winter E, Gustin MC. An osmosensing signal transduction pathway in yeast. Science. 1993 Mar 19;259(5102):1760–1763. [PubMed]
  • Buss F, Stewart M. Macromolecular interactions in the nucleoporin p62 complex of rat nuclear pores: binding of nucleoporin p54 to the rod domain of p62. J Cell Biol. 1995 Feb;128(3):251–261. [PMC free article] [PubMed]
  • Chowdhury S, Smith KW, Gustin MC. Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation. J Cell Biol. 1992 Aug;118(3):561–571. [PMC free article] [PubMed]
  • Copeland CS, Snyder M. Nuclear pore complex antigens delineate nuclear envelope dynamics in vegetative and conjugating Saccharomyces cerevisiae. Yeast. 1993 Mar;9(3):235–249. [PubMed]
  • Costigan C, Gehrung S, Snyder M. A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth. Mol Cell Biol. 1992 Mar;12(3):1162–1178. [PMC free article] [PubMed]
  • Davis LI. The nuclear pore complex. Annu Rev Biochem. 1995;64:865–896. [PubMed]
  • Davis LI, Blobel G. Identification and characterization of a nuclear pore complex protein. Cell. 1986 Jun 6;45(5):699–709. [PubMed]
  • Davis LI, Fink GR. The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell. 1990 Jun 15;61(6):965–978. [PubMed]
  • Doye V, Hurt EC. Genetic approaches to nuclear pore structure and function. Trends Genet. 1995 Jun;11(6):235–241. [PubMed]
  • Doye V, Wepf R, Hurt EC. A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. EMBO J. 1994 Dec 15;13(24):6062–6075. [PubMed]
  • Dujon B, Alexandraki D, André B, Ansorge W, Baladron V, Ballesta JP, Banrevi A, Bolle PA, Bolotin-Fukuhara M, Bossier P, et al. Complete DNA sequence of yeast chromosome XI. Nature. 1994 Jun 2;369(6479):371–378. [PubMed]
  • Fabre E, Boelens WC, Wimmer C, Mattaj IW, Hurt EC. Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif. Cell. 1994 Jul 29;78(2):275–289. [PubMed]
  • Featherstone C, Darby MK, Gerace L. A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J Cell Biol. 1988 Oct;107(4):1289–1297. [PMC free article] [PubMed]
  • Finlay DR, Meier E, Bradley P, Horecka J, Forbes DJ. A complex of nuclear pore proteins required for pore function. J Cell Biol. 1991 Jul;114(1):169–183. [PMC free article] [PubMed]
  • Forrester W, Stutz F, Rosbash M, Wickens M. Defects in mRNA 3'-end formation, transcription initiation, and mRNA transport associated with the yeast mutation prp20: possible coupling of mRNA processing and chromatin structure. Genes Dev. 1992 Oct;6(10):1914–1926. [PubMed]
  • Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. [PubMed]
  • Goldberg MW, Allen TD. High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores. J Cell Biol. 1992 Dec;119(6):1429–1440. [PMC free article] [PubMed]
  • Gorsch LC, Dockendorff TC, Cole CN. A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes. J Cell Biol. 1995 May;129(4):939–955. [PMC free article] [PubMed]
  • Grandi P, Doye V, Hurt EC. Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96. EMBO J. 1993 Aug;12(8):3061–3071. [PubMed]
  • Grandi P, Emig S, Weise C, Hucho F, Pohl T, Hurt EC. A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p. J Cell Biol. 1995 Sep;130(6):1263–1273. [PMC free article] [PubMed]
  • Grandi P, Schlaich N, Tekotte H, Hurt EC. Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p. EMBO J. 1995 Jan 3;14(1):76–87. [PubMed]
  • Hinshaw JE, Carragher BO, Milligan RA. Architecture and design of the nuclear pore complex. Cell. 1992 Jun 26;69(7):1133–1141. [PubMed]
  • Hurwitz ME, Blobel G. NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export. J Cell Biol. 1995 Sep;130(6):1275–1281. [PMC free article] [PubMed]
  • Jarnik M, Aebi U. Toward a more complete 3-D structure of the nuclear pore complex. J Struct Biol. 1991 Dec;107(3):291–308. [PubMed]
  • Kadowaki T, Zhao Y, Tartakoff AM. A conditional yeast mutant deficient in mRNA transport from nucleus to cytoplasm. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2312–2316. [PubMed]
  • Kadowaki T, Chen S, Hitomi M, Jacobs E, Kumagai C, Liang S, Schneiter R, Singleton D, Wisniewska J, Tartakoff AM. Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants. J Cell Biol. 1994 Aug;126(3):649–659. [PMC free article] [PubMed]
  • Kraemer D, Wozniak RW, Blobel G, Radu A. The human CAN protein, a putative oncogene product associated with myeloid leukemogenesis, is a nuclear pore complex protein that faces the cytoplasm. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1519–1523. [PubMed]
  • Kraemer DM, Strambio-de-Castillia C, Blobel G, Rout MP. 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. J Biol Chem. 1995 Aug 11;270(32):19017–19021. [PubMed]
  • Li O, Heath CV, Amberg DC, Dockendorff TC, Copeland CS, Snyder M, Cole CN. Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutive clustering of nuclear pore complexes. Mol Biol Cell. 1995 Apr;6(4):401–417. [PMC free article] [PubMed]
  • Loeb JD, Davis LI, Fink GR. NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex. Mol Biol Cell. 1993 Feb;4(2):209–222. [PMC free article] [PubMed]
  • Maul GG. The nuclear and the cytoplasmic pore complex: structure, dynamics, distribution, and evolution. Int Rev Cytol Suppl. 1977;(6):75–186. [PubMed]
  • Megnet R. Osmotic remedial and osmotic sensitive mutants of Schizosaccharomyces pombe. Experientia. 1966 Apr 15;22(4):216–218. [PubMed]
  • Melchior F, Paschal B, Evans J, Gerace L. Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol. 1993 Dec;123(6 Pt 2):1649–1659. [PMC free article] [PubMed]
  • Metzenberg RL. Repair of multiple defects of a regulatory mutant of Neurospora by high osmotic pressure and by reversion. Arch Biochem Biophys. 1968 May;125(2):532–541. [PubMed]
  • Mirzayan C, Copeland CS, Snyder M. The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton. J Cell Biol. 1992 Mar;116(6):1319–1332. [PMC free article] [PubMed]
  • Moore MS, Blobel G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature. 1993 Oct 14;365(6447):661–663. [PubMed]
  • Moreland RB, Langevin GL, Singer RH, Garcea RL, Hereford LM. Amino acid sequences that determine the nuclear localization of yeast histone 2B. Mol Cell Biol. 1987 Nov;7(11):4048–4057. [PMC free article] [PubMed]
  • Mortimer RK, Contopoulou CR, King JS. Genetic and physical maps of Saccharomyces cerevisiae, Edition 11. Yeast. 1992 Oct;8(10):817–902. [PubMed]
  • Nehrbass U, Kern H, Mutvei A, Horstmann H, Marshallsay B, Hurt EC. NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Cell. 1990 Jun 15;61(6):979–989. [PubMed]
  • Panté N, Bastos R, McMorrow I, Burke B, Aebi U. Interactions and three-dimensional localization of a group of nuclear pore complex proteins. J Cell Biol. 1994 Aug;126(3):603–617. [PMC free article] [PubMed]
  • Park MK, D'Onofrio M, Willingham MC, Hanover JA. A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6462–6466. [PubMed]
  • Pemberton LF, Rout MP, Blobel G. Disruption of the nucleoporin gene NUP133 results in clustering of nuclear pore complexes. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1187–1191. [PubMed]
  • Powers MA, Macaulay C, Masiarz FR, Forbes DJ. Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication. J Cell Biol. 1995 Mar;128(5):721–736. [PMC free article] [PubMed]
  • Radu A, Moore MS, Blobel G. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell. 1995 Apr 21;81(2):215–222. [PubMed]
  • Reed RH, Chudek JA, Foster R, Gadd GM. Osmotic significance of glycerol accumulation in exponentially growing yeasts. Appl Environ Microbiol. 1987 Sep;53(9):2119–2123. [PMC free article] [PubMed]
  • Reichelt R, Holzenburg A, Buhle EL, Jr, Jarnik M, Engel A, Aebi U. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol. 1990 Apr;110(4):883–894. [PMC free article] [PubMed]
  • Ren M, Drivas G, D'Eustachio P, Rush MG. Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis. J Cell Biol. 1993 Jan;120(2):313–323. [PMC free article] [PubMed]
  • Richardson WD, Mills AD, Dilworth SM, Laskey RA, Dingwall C. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell. 1988 Mar 11;52(5):655–664. [PubMed]
  • Riles L, Dutchik JE, Baktha A, McCauley BK, Thayer EC, Leckie MP, Braden VV, Depke JE, Olson MV. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. [PubMed]
  • Ris H, Malecki M. High-resolution field emission scanning electron microscope imaging of internal cell structures after Epon extraction from sections: a new approach to correlative ultrastructural and immunocytochemical studies. J Struct Biol. 1993 Sep-Oct;111(2):148–157. [PubMed]
  • Rout MP, Blobel G. Isolation of the yeast nuclear pore complex. J Cell Biol. 1993 Nov;123(4):771–783. [PMC free article] [PubMed]
  • Rout MP, Wente SR. Pores for thought: nuclear pore complex proteins. Trends Cell Biol. 1994 Oct;4(10):357–365. [PubMed]
  • Schlenstedt G, Saavedra C, Loeb JD, Cole CN, Silver PA. The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):225–229. [PubMed]
  • Severs NJ, Jordan EG, Williamson DH. Nuclear pore absence from areas of close association between nucleus and vacuole in synchronous yeast cultures. J Ultrastruct Res. 1976 Mar;54(3):374–387. [PubMed]
  • Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. [PubMed]
  • Snow CM, Senior A, Gerace L. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol. 1987 May;104(5):1143–1156. [PMC free article] [PubMed]
  • Tollervey D, Mattaj IW. Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs. EMBO J. 1987 Feb;6(2):469–476. [PubMed]
  • Traglia HM, Atkinson NS, Hopper AK. Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes. Mol Cell Biol. 1989 Jul;9(7):2989–2999. [PMC free article] [PubMed]
  • Udem SA, Warner JR. The cytoplasmic maturation of a ribosomal precursor ribonucleic acid in yeast. J Biol Chem. 1973 Feb 25;248(4):1412–1416. [PubMed]
  • Unwin PN, Milligan RA. A large particle associated with the perimeter of the nuclear pore complex. J Cell Biol. 1982 Apr;93(1):63–75. [PMC free article] [PubMed]
  • Varela JC, van Beekvelt C, Planta RJ, Mager WH. Osmostress-induced changes in yeast gene expression. Mol Microbiol. 1992 Aug;6(15):2183–2190. [PubMed]
  • Wente SR, Blobel G. A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic. J Cell Biol. 1993 Oct;123(2):275–284. [PMC free article] [PubMed]
  • Wente SR, Blobel G. NUP145 encodes a novel yeast glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin required for nuclear envelope structure. J Cell Biol. 1994 Jun;125(5):955–969. [PMC free article] [PubMed]
  • Wente SR, Rout MP, Blobel G. A new family of yeast nuclear pore complex proteins. J Cell Biol. 1992 Nov;119(4):705–723. [PMC free article] [PubMed]
  • Wimmer C, Doye V, Grandi P, Nehrbass U, Hurt EC. A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1. EMBO J. 1992 Dec;11(13):5051–5061. [PubMed]
  • Wozniak RW, Blobel G, Rout MP. POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope. J Cell Biol. 1994 Apr;125(1):31–42. [PMC free article] [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press