Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1999 Jan;151(1):57–75. doi: 10.1093/genetics/151.1.57

Saccharomyces cerevisiae Mod5p-II contains sequences antagonistic for nuclear and cytosolic locations.

L H Tolerico 1, A L Benko 1, J P Aris 1, D R Stanford 1, N C Martin 1, A K Hopper 1
PMCID: PMC1460473  PMID: 9872948

Abstract

MOD5 encodes a tRNA modification activity located in three subcellular compartments. Alternative translation initiation generates Mod5p-I, located in the mitochondria and the cytosol, and Mod5p-II, located in the cytosol and nucleus. Here we study the nucleus/cytosol distribution of overexpressed Mod5p-II. Nuclear Mod5p-II appears concentrated in the nucleolus, perhaps indicating that the nuclear pool may have a different biological role than the cytoplasmic and mitochondrial pools. Mod5p contains three motifs resembling bipartite-like nuclear localization sequences (NLSs), but only one is sufficient to locate a passenger protein to the nucleus. Mutations of basic residues of this motif cumulatively contribute to a cytosolic location for the fusion proteins. These alterations also cause decreased nuclear pools of endogenous Mod5p-II. Depletion of nuclear Mod5p-II does not affect tRNATyr function. Despite the NLS, most Mod5p is cytosolic. We assessed whether Mod5p sequences cause a karyophilic reporter to be located in the cytosol. By this assay, Mod5p may contain more than one region that functions as cytoplasmic retention and/or nuclear export sequences. Thus, distribution of Mod5p results from the presence/absence of mitochondrial targeting information and sequences antagonistic for nuclear and cytosolic locations. Mod5p is highly conserved; sequences responsible for subcellular distribution appear to reside in "accessory" motifs missing from prokaryotic counterparts.

Full Text

The Full Text of this article is available as a PDF (572.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aris J. P., 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. doi: 10.1083/jcb.107.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benton B. M., Eng W. K., Dunn J. J., Studier F. W., Sternglanz R., Fisher P. A. Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes. Mol Cell Biol. 1990 Jan;10(1):353–360. doi: 10.1128/mcb.10.1.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertrand E., Houser-Scott F., Kendall A., Singer R. H., Engelke D. R. Nucleolar localization of early tRNA processing. Genes Dev. 1998 Aug 15;12(16):2463–2468. doi: 10.1101/gad.12.16.2463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boguta M., Hunter L. A., Shen W. C., Gillman E. C., Martin N. C., Hopper A. K. Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol. Mol Cell Biol. 1994 Apr;14(4):2298–2306. doi: 10.1128/mcb.14.4.2298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chamberlain J. R., Pagán-Ramos, Kindelberger D. W., Engelke D. R. An RNase P RNA subunit mutation affects ribosomal RNA processing. Nucleic Acids Res. 1996 Aug 15;24(16):3158–3166. doi: 10.1093/nar/24.16.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen D. C., Yang B. C., Kuo T. T. One-step transformation of yeast in stationary phase. Curr Genet. 1992 Jan;21(1):83–84. doi: 10.1007/BF00318659. [DOI] [PubMed] [Google Scholar]
  8. Chen S., Brockenbrough J. S., Dove J. E., Aris J. P. Homocitrate synthase is located in the nucleus in the yeast Saccharomyces cerevisiae. J Biol Chem. 1997 Apr 18;272(16):10839–10846. doi: 10.1074/jbc.272.16.10839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. doi: 10.1016/0378-1119(92)90454-w. [DOI] [PubMed] [Google Scholar]
  10. Clark M. W., Abelson J. The subnuclear localization of tRNA ligase in yeast. J Cell Biol. 1987 Oct;105(4):1515–1526. doi: 10.1083/jcb.105.4.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Danpure C. J. How can the products of a single gene be localized to more than one intracellular compartment? Trends Cell Biol. 1995 Jun;5(6):230–238. doi: 10.1016/s0962-8924(00)89016-9. [DOI] [PubMed] [Google Scholar]
  12. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  13. Dove J. E., Brockenbrough J. S., Aris J. P. Isolation of nuclei and nucleoli from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 1998;53:33–46. doi: 10.1016/s0091-679x(08)60873-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fischer U., Huber J., Boelens W. C., Mattaj I. W., Lührmann R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell. 1995 Aug 11;82(3):475–483. doi: 10.1016/0092-8674(95)90436-0. [DOI] [PubMed] [Google Scholar]
  15. Gerace L. Nuclear export signals and the fast track to the cytoplasm. Cell. 1995 Aug 11;82(3):341–344. doi: 10.1016/0092-8674(95)90420-4. [DOI] [PubMed] [Google Scholar]
  16. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gillman E. C., Slusher L. B., Martin N. C., Hopper A. K. MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA. Mol Cell Biol. 1991 May;11(5):2382–2390. doi: 10.1128/mcb.11.5.2382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanahan D., Jessee J., Bloom F. R. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 1991;204:63–113. doi: 10.1016/0076-6879(91)04006-a. [DOI] [PubMed] [Google Scholar]
  19. Henríquez R., Blobel G., Aris J. P. Isolation and sequencing of NOP1. A yeast gene encoding a nucleolar protein homologous to a human autoimmune antigen. J Biol Chem. 1990 Feb 5;265(4):2209–2215. [PubMed] [Google Scholar]
  20. Higgins D. G., Thompson J. D., Gibson T. J. Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 1996;266:383–402. doi: 10.1016/s0076-6879(96)66024-8. [DOI] [PubMed] [Google Scholar]
  21. Hopper A. K., Furukawa A. H., Pham H. D., Martin N. C. Defects in modification of cytoplasmic and mitochondrial transfer RNAs are caused by single nuclear mutations. Cell. 1982 Mar;28(3):543–550. doi: 10.1016/0092-8674(82)90209-4. [DOI] [PubMed] [Google Scholar]
  22. Hopper A. K., Traglia H. M., Dunst R. W. The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus. J Cell Biol. 1990 Aug;111(2):309–321. doi: 10.1083/jcb.111.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hurt E. C. A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae. EMBO J. 1988 Dec 20;7(13):4323–4334. doi: 10.1002/j.1460-2075.1988.tb03331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
  25. Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  27. Makkerh J. P., Dingwall C., Laskey R. A. Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids. Curr Biol. 1996 Aug 1;6(8):1025–1027. doi: 10.1016/s0960-9822(02)00648-6. [DOI] [PubMed] [Google Scholar]
  28. Michael W. M., Choi M., Dreyfuss G. A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell. 1995 Nov 3;83(3):415–422. doi: 10.1016/0092-8674(95)90119-1. [DOI] [PubMed] [Google Scholar]
  29. Michael W. M., Eder P. S., Dreyfuss G. The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein. EMBO J. 1997 Jun 16;16(12):3587–3598. doi: 10.1093/emboj/16.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moreland R. B., Langevin G. L., Singer R. H., Garcea R. L., Hereford L. M. Amino acid sequences that determine the nuclear localization of yeast histone 2B. Mol Cell Biol. 1987 Nov;7(11):4048–4057. doi: 10.1128/mcb.7.11.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murphy R., Wente S. R. An RNA-export mediator with an essential nuclear export signal. Nature. 1996 Sep 26;383(6598):357–360. doi: 10.1038/383357a0. [DOI] [PubMed] [Google Scholar]
  32. Najarian D., Dihanich M. E., Martin N. C., Hopper A. K. DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts. Mol Cell Biol. 1987 Jan;7(1):185–191. doi: 10.1128/mcb.7.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nishikura K., De Robertis E. M. RNA processing in microinjected Xenopus oocytes. Sequential addition of base modifications in the spliced transfer RNA. J Mol Biol. 1981 Jan 15;145(2):405–420. doi: 10.1016/0022-2836(81)90212-6. [DOI] [PubMed] [Google Scholar]
  34. Peebles C. L., Gegenheimer P., Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. doi: 10.1016/0092-8674(83)90472-5. [DOI] [PubMed] [Google Scholar]
  35. Pines J., Hunter T. The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J. 1994 Aug 15;13(16):3772–3781. doi: 10.1002/j.1460-2075.1994.tb06688.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  37. Rose A. M., Belford H. G., Shen W. C., Greer C. L., Hopper A. K., Martin N. C. Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase. Biochimie. 1995;77(1-2):45–53. doi: 10.1016/0300-9084(96)88103-x. [DOI] [PubMed] [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shou W., Li X., Wu C., Cao T., Kuang J., Che S., Etkin L. D. Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues. Mol Cell Biol. 1996 Mar;16(3):990–997. doi: 10.1128/mcb.16.3.990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tollervey D., Kiss T. Function and synthesis of small nucleolar RNAs. Curr Opin Cell Biol. 1997 Jun;9(3):337–342. doi: 10.1016/s0955-0674(97)80005-1. [DOI] [PubMed] [Google Scholar]
  42. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  43. Wolfe C. L., Hopper A. K., Martin N. C. Mechanisms leading to and the consequences of altering the normal distribution of ATP(CTP):tRNA nucleotidyltransferase in yeast. J Biol Chem. 1996 Mar 1;271(9):4679–4686. doi: 10.1074/jbc.271.9.4679. [DOI] [PubMed] [Google Scholar]
  44. Wolfe C. L., Lou Y. C., Hopper A. K., Martin N. C. Interplay of heterogeneous transcriptional start sites and translational selection of AUGs dictate the production of mitochondrial and cytosolic/nuclear tRNA nucleotidyltransferase from the same gene in yeast. J Biol Chem. 1994 May 6;269(18):13361–13366. [PubMed] [Google Scholar]
  45. Zoladek T., Vaduva G., Hunter L. A., Boguta M., Go B. D., Martin N. C., Hopper A. K. Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery. Mol Cell Biol. 1995 Dec;15(12):6884–6894. doi: 10.1128/mcb.15.12.6884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. de Beus E., Brockenbrough J. S., Hong B., Aris J. P. Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker. J Cell Biol. 1994 Dec;127(6 Pt 2):1799–1813. doi: 10.1083/jcb.127.6.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 1986 Jun;5(6):1335–1342. doi: 10.1002/j.1460-2075.1986.tb04364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES