Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2000 Jun;155(2):611–622. doi: 10.1093/genetics/155.2.611

Loss of Rhb1, a Rheb-related GTPase in fission yeast, causes growth arrest with a terminal phenotype similar to that caused by nitrogen starvation.

K E Mach 1, K A Furge 1, C F Albright 1
PMCID: PMC1461131  PMID: 10835385

Abstract

The Rheb GTPase is most similar in primary sequence to the Ras, Rap, R-Ras, and Ral GTPases, which regulate cell growth and differentiation in many cell types. A likely fission yeast homologue of mammalian Rheb, which we designated Rhb1, was identified by genome sequencing. Our investigation of rhb1 showed that rhb1(-) cells arrested cell growth and division with a terminal phenotype similar to that of nitrogen-starved cells. In particular, cells depleted of Rhb1 arrested as small, round cells with 1N DNA content, arrested more quickly in low-nitrogen medium, and induced expression of fnx1 and mei2 mRNA, two mRNAs that were normally induced by nitrogen starvation. Since mammalian Rheb binds and may regulate Raf-1, a Ras effector, we tested for functional overlap between Ras1 and Rhb1 in fission yeast. This analysis showed that Ras1 overexpression did not suppress rhb1(-) mutant phenotypes, Rhb1 overexpression did not suppress ras1(-) mutant phenotypes, and ras1(-) rhb1(-) double mutants had phenotypes equal to the sum of the corresponding single-mutant phenotypes. Hence, there is no evidence for overlapping functions between Ras1 and Rhb1. On the basis of this study, we hypothesize that Rhb1 negatively regulates entry into stationary phase when extracellular nitrogen levels are adequate for growth. If this hypothesis is correct, then Rhb1 and Ras1 regulate alternative responses to limiting nutrients.

Full Text

The Full Text of this article is available as a PDF (551.8 KB).

Selected References

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

  1. Basi G., Schmid E., Maundrell K. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene. 1993 Jan 15;123(1):131–136. doi: 10.1016/0378-1119(93)90552-e. [DOI] [PubMed] [Google Scholar]
  2. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  3. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  4. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  5. Campbell S. L., Khosravi-Far R., Rossman K. L., Clark G. J., Der C. J. Increasing complexity of Ras signaling. Oncogene. 1998 Sep 17;17(11 REVIEWS):1395–1413. doi: 10.1038/sj.onc.1202174. [DOI] [PubMed] [Google Scholar]
  6. Chang E. C., Barr M., Wang Y., Jung V., Xu H. P., Wigler M. H. Cooperative interaction of S. pombe proteins required for mating and morphogenesis. Cell. 1994 Oct 7;79(1):131–141. doi: 10.1016/0092-8674(94)90406-5. [DOI] [PubMed] [Google Scholar]
  7. Clark G. J., Kinch M. S., Rogers-Graham K., Sebti S. M., Hamilton A. D., Der C. J. The Ras-related protein Rheb is farnesylated and antagonizes Ras signaling and transformation. J Biol Chem. 1997 Apr 18;272(16):10608–10615. doi: 10.1074/jbc.272.16.10608. [DOI] [PubMed] [Google Scholar]
  8. Cook S. J., McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science. 1993 Nov 12;262(5136):1069–1072. doi: 10.1126/science.7694367. [DOI] [PubMed] [Google Scholar]
  9. DeVoti J., Seydoux G., Beach D., McLeod M. Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis. EMBO J. 1991 Dec;10(12):3759–3768. doi: 10.1002/j.1460-2075.1991.tb04945.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dimitrov K., Sazer S. The role of fnx1, a fission yeast multidrug resistance protein, in the transition of cells to a quiescent G0 state. Mol Cell Biol. 1998 Sep;18(9):5239–5246. doi: 10.1128/mcb.18.9.5239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fantes P., Nurse P. Control of cell size at division in fission yeast by a growth-modulated size control over nuclear division. Exp Cell Res. 1977 Jul;107(2):377–386. doi: 10.1016/0014-4827(77)90359-7. [DOI] [PubMed] [Google Scholar]
  12. Fisher D. L., Nurse P. A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins. EMBO J. 1996 Feb 15;15(4):850–860. [PMC free article] [PubMed] [Google Scholar]
  13. Forsburg S. L. Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res. 1993 Jun 25;21(12):2955–2956. doi: 10.1093/nar/21.12.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fukui Y., Kozasa T., Kaziro Y., Takeda T., Yamamoto M. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell. 1986 Jan 31;44(2):329–336. doi: 10.1016/0092-8674(86)90767-1. [DOI] [PubMed] [Google Scholar]
  15. Fukui Y., Yamamoto M. Isolation and characterization of Schizosaccharomyces pombe mutants phenotypically similar to ras1-. Mol Gen Genet. 1988 Dec;215(1):26–31. doi: 10.1007/BF00331298. [DOI] [PubMed] [Google Scholar]
  16. Gromov P. S., Madsen P., Tomerup N., Celis J. E. A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of rheb. FEBS Lett. 1995 Dec 18;377(2):221–226. doi: 10.1016/0014-5793(95)01349-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mach K. E., Cheng Q., Albright C. F. ras1 and pat1 alleles interact to quantitatively and qualitatively alter conjugation in fission yeast. Curr Genet. 1998 Sep;34(3):172–182. doi: 10.1007/s002940050383. [DOI] [PubMed] [Google Scholar]
  20. Maeda T., Mochizuki N., Yamamoto M. Adenylyl cyclase is dispensable for vegetative cell growth in the fission yeast Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7814–7818. doi: 10.1073/pnas.87.20.7814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Masuda T., Kariya K., Shinkai M., Okada T., Kataoka T. Protein kinase Byr2 is a target of Ras1 in the fission yeast Schizosaccharomyces pombe. J Biol Chem. 1995 Feb 3;270(5):1979–1982. doi: 10.1074/jbc.270.5.1979. [DOI] [PubMed] [Google Scholar]
  22. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  23. Millar J. B., Buck V., Wilkinson M. G. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast. Genes Dev. 1995 Sep 1;9(17):2117–2130. doi: 10.1101/gad.9.17.2117. [DOI] [PubMed] [Google Scholar]
  24. Mochizuki N., Yamamoto M. Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast. Mol Gen Genet. 1992 May;233(1-2):17–24. doi: 10.1007/BF00587556. [DOI] [PubMed] [Google Scholar]
  25. Moores S. L., Schaber M. D., Mosser S. D., Rands E., O'Hara M. B., Garsky V. M., Marshall M. S., Pompliano D. L., Gibbs J. B. Sequence dependence of protein isoprenylation. J Biol Chem. 1991 Aug 5;266(22):14603–14610. [PubMed] [Google Scholar]
  26. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  27. Nadin-Davis S. A., Nasim A. A gene which encodes a predicted protein kinase can restore some functions of the ras gene in fission yeast. EMBO J. 1988 Apr;7(4):985–993. doi: 10.1002/j.1460-2075.1988.tb02905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nadin-Davis S. A., Nasim A., Beach D. Involvement of ras in sexual differentiation but not in growth control in fission yeast. EMBO J. 1986 Nov;5(11):2963–2971. doi: 10.1002/j.1460-2075.1986.tb04593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shiozaki K., Russell P. Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature. 1995 Dec 14;378(6558):739–743. doi: 10.1038/378739a0. [DOI] [PubMed] [Google Scholar]
  30. Shiozaki K., Russell P. Conjugation, meiosis, and the osmotic stress response are regulated by Spc1 kinase through Atf1 transcription factor in fission yeast. Genes Dev. 1996 Sep 15;10(18):2276–2288. doi: 10.1101/gad.10.18.2276. [DOI] [PubMed] [Google Scholar]
  31. Shirayama M., Matsui Y., Toh-E A. The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase. Mol Cell Biol. 1994 Nov;14(11):7476–7482. doi: 10.1128/mcb.14.11.7476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Song K., Mach K. E., Chen C. Y., Reynolds T., Albright C. F. A novel suppressor of ras1 in fission yeast, byr4, is a dosage-dependent inhibitor of cytokinesis. J Cell Biol. 1996 Jun;133(6):1307–1319. doi: 10.1083/jcb.133.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stang S., Bottorff D., Stone J. C. Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells. Mol Cell Biol. 1997 Jun;17(6):3047–3055. doi: 10.1128/mcb.17.6.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Su S. S., Tanaka Y., Samejima I., Tanaka K., Yanagida M. A nitrogen starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted G1 state and its delay for return to proliferation. J Cell Sci. 1996 Jun;109(Pt 6):1347–1357. doi: 10.1242/jcs.109.6.1347. [DOI] [PubMed] [Google Scholar]
  35. Toda T., Shimanuki M., Yanagida M. Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. Genes Dev. 1991 Jan;5(1):60–73. doi: 10.1101/gad.5.1.60. [DOI] [PubMed] [Google Scholar]
  36. Tu H., Barr M., Dong D. L., Wigler M. Multiple regulatory domains on the Byr2 protein kinase. Mol Cell Biol. 1997 Oct;17(10):5876–5887. doi: 10.1128/mcb.17.10.5876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Van Aelst L., Barr M., Marcus S., Polverino A., Wigler M. Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6213–6217. doi: 10.1073/pnas.90.13.6213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wang Y., Xu H. P., Riggs M., Rodgers L., Wigler M. byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype. Mol Cell Biol. 1991 Jul;11(7):3554–3563. doi: 10.1128/mcb.11.7.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Werner-Washburne M., Braun E., Johnston G. C., Singer R. A. Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol Rev. 1993 Jun;57(2):383–401. doi: 10.1128/mr.57.2.383-401.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wu J., Dent P., Jelinek T., Wolfman A., Weber M. J., Sturgill T. W. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science. 1993 Nov 12;262(5136):1065–1069. doi: 10.1126/science.7694366. [DOI] [PubMed] [Google Scholar]
  41. Yee W. M., Worley P. F. Rheb interacts with Raf-1 kinase and may function to integrate growth factor- and protein kinase A-dependent signals. Mol Cell Biol. 1997 Feb;17(2):921–933. doi: 10.1128/mcb.17.2.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Young D., Riggs M., Field J., Vojtek A., Broek D., Wigler M. The adenylyl cyclase gene from Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7989–7993. doi: 10.1073/pnas.86.20.7989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES