Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Apr;14(4):2391–2403. doi: 10.1128/mcb.14.4.2391

NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway.

C Costigan 1, D Kolodrubetz 1, M Snyder 1
PMCID: PMC358606  PMID: 8139543

Abstract

The yeast SLK1 (BCK1) gene encodes a mitogen-activated protein kinase (MAPK) activator protein which functions upstream in a protein kinase cascade that converges on the MAPK Slt2p (Mpk1p). Dominant alleles of SLK1 have been shown to bypass the conditional lethality of a protein kinase C mutation, pkc1-delta, suggesting that Pkc1p may regulate Slk1p function. Slk1p has an important role in morphogenesis and growth control, and deletions of the SLK1 gene are lethal in a spa2-delta mutant background. To search for genes that interact with the SLK1-SLT2 pathway, a synthetic lethal suppression screen was carried out. Genes which in multiple copies suppress the synthetic lethality of slk1-1 spa2-delta were identified, and one, the NHP6A gene, has been extensively characterized. The NHP6A gene and the closely related NHP6B gene were shown previously to encode HMG1-like chromatin-associated proteins. We demonstrate here that these genes are functionally redundant and that multiple copies of either NHP6A or NHP6B suppress slk1-delta and slt2-delta. Strains from which both NHP6 genes were deleted (nhp6-delta mutants) share many phenotypes with pkc1-delta, slk1-delta, and slt2-delta mutants. nhp6-delta cells display a temperature-sensitive growth defect that is rescued by the addition of 1 M sorbitol to the medium, and they are sensitive to starvation. nhp6-delta strains also exhibit a variety of morphological and cytoskeletal defects. At the restrictive temperature for growth, nhp6-delta mutant cells contain elongated buds and enlarged necks. Many cells have patches of chitin staining on their cell surfaces, and chitin deposition is enhanced at the necks of budded cells. nhp6-delta cells display a defect in actin polarity and often accumulate large actin chunks. Genetic and phenotypic analysis indicates that NHP6A and NHP6B function downstream of SLT2. Our results indicate that the Slt2p MAPK pathway in Saccharomyces cerevisiae may mediate its function in cell growth and morphogenesis, at least in part, through high-mobility group proteins.

Full text

PDF
2391

Images in this article

2395Image
on p.2395
2396Image
on p.2396
2398Image
on p.2398
2399Image
on p.2399
2400Image
on p.2400

Selected References

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

  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahn N. G., Seger R., Krebs E. G. The mitogen-activated protein kinase activator. Curr Opin Cell Biol. 1992 Dec;4(6):992–999. doi: 10.1016/0955-0674(92)90131-u. [DOI] [PubMed] [Google Scholar]
  3. Alvarez E., Northwood I. C., Gonzalez F. A., Latour D. A., Seth A., Abate C., Curran T., Davis R. J. Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. J Biol Chem. 1991 Aug 15;266(23):15277–15285. [PubMed] [Google Scholar]
  4. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5889–5892. doi: 10.1073/pnas.90.13.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  6. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  7. Brewster J. L., de Valoir T., Dwyer N. D., Winter E., Gustin M. C. An osmosensing signal transduction pathway in yeast. Science. 1993 Mar 19;259(5102):1760–1763. doi: 10.1126/science.7681220. [DOI] [PubMed] [Google Scholar]
  8. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  9. Chen R. H., Sarnecki C., Blenis J. Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol. 1992 Mar;12(3):915–927. doi: 10.1128/mcb.12.3.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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. doi: 10.1128/mcb.12.3.1162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Courchesne W. E., Kunisawa R., Thorner J. A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae. Cell. 1989 Sep 22;58(6):1107–1119. doi: 10.1016/0092-8674(89)90509-6. [DOI] [PubMed] [Google Scholar]
  12. Dent P., Haser W., Haystead T. A., Vincent L. A., Roberts T. M., Sturgill T. W. Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science. 1992 Sep 4;257(5075):1404–1407. doi: 10.1126/science.1326789. [DOI] [PubMed] [Google Scholar]
  13. Elion E. A., Grisafi P. L., Fink G. R. FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation. Cell. 1990 Feb 23;60(4):649–664. doi: 10.1016/0092-8674(90)90668-5. [DOI] [PubMed] [Google Scholar]
  14. Engebrecht J., Hirsch J., Roeder G. S. Meiotic gene conversion and crossing over: their relationship to each other and to chromosome synapsis and segregation. Cell. 1990 Sep 7;62(5):927–937. doi: 10.1016/0092-8674(90)90267-i. [DOI] [PubMed] [Google Scholar]
  15. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  16. Flescher E. G., Madden K., Snyder M. Components required for cytokinesis are important for bud site selection in yeast. J Cell Biol. 1993 Jul;122(2):373–386. doi: 10.1083/jcb.122.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gartner A., Nasmyth K., Ammerer G. Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1. Genes Dev. 1992 Jul;6(7):1280–1292. doi: 10.1101/gad.6.7.1280. [DOI] [PubMed] [Google Scholar]
  18. Gehrung S., Snyder M. The SPA2 gene of Saccharomyces cerevisiae is important for pheromone-induced morphogenesis and efficient mating. J Cell Biol. 1990 Oct;111(4):1451–1464. doi: 10.1083/jcb.111.4.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gille H., Sharrocks A. D., Shaw P. E. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Nature. 1992 Jul 30;358(6385):414–417. doi: 10.1038/358414a0. [DOI] [PubMed] [Google Scholar]
  20. Gimeno C. J., Ljungdahl P. O., Styles C. A., Fink G. R. Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell. 1992 Mar 20;68(6):1077–1090. doi: 10.1016/0092-8674(92)90079-r. [DOI] [PubMed] [Google Scholar]
  21. Golemis E. A., Brent R. Fused protein domains inhibit DNA binding by LexA. Mol Cell Biol. 1992 Jul;12(7):3006–3014. doi: 10.1128/mcb.12.7.3006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gómez N., Cohen P. Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature. 1991 Sep 12;353(6340):170–173. doi: 10.1038/353170a0. [DOI] [PubMed] [Google Scholar]
  23. Haycock J. W., Ahn N. G., Cobb M. H., Krebs E. G. ERK1 and ERK2, two microtubule-associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2365–2369. doi: 10.1073/pnas.89.6.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  25. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  26. Holtzman D. A., Yang S., Drubin D. G. Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J Cell Biol. 1993 Aug;122(3):635–644. doi: 10.1083/jcb.122.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Irie K., Takase M., Lee K. S., Levin D. E., Araki H., Matsumoto K., Oshima Y. MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C. Mol Cell Biol. 1993 May;13(5):3076–3083. doi: 10.1128/mcb.13.5.3076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jackson J. B., Pollock J. M., Jr, Rill R. L. Chromatin fractionation procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins. Biochemistry. 1979 Aug 21;18(17):3739–3748. doi: 10.1021/bi00584a015. [DOI] [PubMed] [Google Scholar]
  29. Kolodrubetz D., Burgum A. Duplicated NHP6 genes of Saccharomyces cerevisiae encode proteins homologous to bovine high mobility group protein 1. J Biol Chem. 1990 Feb 25;265(6):3234–3239. [PubMed] [Google Scholar]
  30. Kolodrubetz D., Haggren W., Burgum A. Amino-terminal sequence of a Saccharomyces cerevisiae nuclear protein, NHP6, shows significant identity to bovine HMG1. FEBS Lett. 1988 Sep 26;238(1):175–179. doi: 10.1016/0014-5793(88)80251-5. [DOI] [PubMed] [Google Scholar]
  31. Kyriakis J. M., App H., Zhang X. F., Banerjee P., Brautigan D. L., Rapp U. R., Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992 Jul 30;358(6385):417–421. doi: 10.1038/358417a0. [DOI] [PubMed] [Google Scholar]
  32. Köhrer K., Domdey H. Preparation of high molecular weight RNA. Methods Enzymol. 1991;194:398–405. doi: 10.1016/0076-6879(91)94030-g. [DOI] [PubMed] [Google Scholar]
  33. Lange-Carter C. A., Pleiman C. M., Gardner A. M., Blumer K. J., Johnson G. L. A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science. 1993 Apr 16;260(5106):315–319. doi: 10.1126/science.8385802. [DOI] [PubMed] [Google Scholar]
  34. Lee K. S., Irie K., Gotoh Y., Watanabe Y., Araki H., Nishida E., Matsumoto K., Levin D. E. A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C. Mol Cell Biol. 1993 May;13(5):3067–3075. doi: 10.1128/mcb.13.5.3067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee K. S., Levin D. E. Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. Mol Cell Biol. 1992 Jan;12(1):172–182. doi: 10.1128/mcb.12.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Levy W B., Wong N. C., Dixon G. H. Selective association of the trout-specific H6 protein with chromatin regions susceptible to DNase I and DNase II: possible location of HMG-T in the spacer region between core nucleosomes. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2810–2814. doi: 10.1073/pnas.74.7.2810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  39. Madden K., Costigan C., Snyder M. Cell polarity and morphogenesis in Saccharomyces cerevisiae. Trends Cell Biol. 1992 Jan;2(1):22–29. doi: 10.1016/0962-8924(92)90140-i. [DOI] [PubMed] [Google Scholar]
  40. Madden K., Snyder M. Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection. Mol Biol Cell. 1992 Sep;3(9):1025–1035. doi: 10.1091/mbc.3.9.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mazzoni C., Zarov P., Rambourg A., Mann C. The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisiae. J Cell Biol. 1993 Dec;123(6 Pt 2):1821–1833. doi: 10.1083/jcb.123.6.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Neiman A. M., Stevenson B. J., Xu H. P., Sprague G. F., Jr, Herskowitz I., Wigler M., Marcus S. Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms. Mol Biol Cell. 1993 Jan;4(1):107–120. doi: 10.1091/mbc.4.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Paravicini G., Cooper M., Friedli L., Smith D. J., Carpentier J. L., Klig L. S., Payton M. A. The osmotic integrity of the yeast cell requires a functional PKC1 gene product. Mol Cell Biol. 1992 Nov;12(11):4896–4905. doi: 10.1128/mcb.12.11.4896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Payne D. M., Rossomando A. J., Martino P., Erickson A. K., Her J. H., Shabanowitz J., Hunt D. F., Weber M. J., Sturgill T. W. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 1991 Apr;10(4):885–892. doi: 10.1002/j.1460-2075.1991.tb08021.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  46. Rossomando A., Wu J., Weber M. J., Sturgill T. W. The phorbol ester-dependent activator of the mitogen-activated protein kinase p42mapk is a kinase with specificity for the threonine and tyrosine regulatory sites. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5221–5225. doi: 10.1073/pnas.89.12.5221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  48. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  49. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Seth A., Gonzalez F. A., Gupta S., Raden D. L., Davis R. J. Signal transduction within the nucleus by mitogen-activated protein kinase. J Biol Chem. 1992 Dec 5;267(34):24796–24804. [PubMed] [Google Scholar]
  52. 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]
  53. Sloat B. F., Pringle J. R. A mutant of yeast defective in cellular morphogenesis. Science. 1978 Jun 9;200(4346):1171–1173. doi: 10.1126/science.349694. [DOI] [PubMed] [Google Scholar]
  54. Snyder M., Gehrung S., Page B. D. Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae. J Cell Biol. 1991 Aug;114(3):515–532. doi: 10.1083/jcb.114.3.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Snyder M. The SPA2 protein of yeast localizes to sites of cell growth. J Cell Biol. 1989 Apr;108(4):1419–1429. doi: 10.1083/jcb.108.4.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sturgill T. W., Ray L. B., Erikson E., Maller J. L. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature. 1988 Aug 25;334(6184):715–718. doi: 10.1038/334715a0. [DOI] [PubMed] [Google Scholar]
  57. Tercero J. C., Riles L. E., Wickner R. B. Localized mutagenesis and evidence for post-transcriptional regulation of MAK3. A putative N-acetyltransferase required for double-stranded RNA virus propagation in Saccharomyces cerevisiae. J Biol Chem. 1992 Oct 5;267(28):20270–20276. [PubMed] [Google Scholar]
  58. Torres L., Martín H., García-Saez M. I., Arroyo J., Molina M., Sánchez M., Nombela C. A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants. Mol Microbiol. 1991 Nov;5(11):2845–2854. doi: 10.1111/j.1365-2958.1991.tb01993.x. [DOI] [PubMed] [Google Scholar]
  59. Tremethick D. J., Molloy P. L. Effects of high mobility group proteins 1 and 2 on initiation and elongation of specific transcription by RNA polymerase II in vitro. Nucleic Acids Res. 1988 Dec 9;16(23):11107–11123. doi: 10.1093/nar/16.23.11107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Warner J. R. Labeling of RNA and phosphoproteins in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:423–428. doi: 10.1016/0076-6879(91)94033-9. [DOI] [PubMed] [Google Scholar]
  61. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  62. Xie K., Lambie E. J., Snyder M. Nuclear dot antigens may specify transcriptional domains in the nucleus. Mol Cell Biol. 1993 Oct;13(10):6170–6179. doi: 10.1128/mcb.13.10.6170. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES