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. 1996 Jan;16(1):168–178. doi: 10.1128/mcb.16.1.168

Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae.

L R Kao 1, J Peterson 1, R Ji 1, L Bender 1, A Bender 1
PMCID: PMC230990  PMID: 8524293

Abstract

Akr1p, which contains six ankyrin repeats, was identified during a screen for mutations that displayed synthetic lethality with a mutant allele of the bud emergence gene BEM1. Cells from which AKR1 had been deleted were alive but misshapen at 30 degrees C and inviable at 37 degrees C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G alpha subunit of the pheromone receptor-coupled G protein. (The active subunit of this G protein is G beta gamma, and G alpha plays an inhibitory role in G beta gamma-mediated signal transduction.) AKR1 could serve as a multicopy suppressor of the lethality caused by either loss of GPA1 or overexpression of STE4, which encodes the G beta subunit of this G protein, suggesting that pheromone signaling is inhibited by overexpression of Akr1p. Mutations in AKR1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just when overexpressed) inhibits signaling. In contrast, deletion of BEM1 resulted in decreased expression of FUS1, suggesting that Bem1p normally facilitates pheromone signaling. During a screen for proteins that displayed two-hybrid interactions with Akr1p, we identified Ste4p, raising the possibility that an interaction between Akr1p and Ste4p contributes to proper regulation of the pheromone response pathway.

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Selected References

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  1. Adams A. E., Johnson D. I., Longnecker R. M., Sloat B. F., Pringle J. R. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol. 1990 Jul;111(1):131–142. doi: 10.1083/jcb.111.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender A., Pringle J. R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. doi: 10.1073/pnas.86.24.9976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bender A., Pringle J. R. 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. doi: 10.1128/mcb.11.3.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bender A., Sprague G. F., Jr Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae. Genetics. 1989 Mar;121(3):463–476. doi: 10.1093/genetics/121.3.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Chant J., Corrado K., Pringle J. R., Herskowitz I. Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1. Cell. 1991 Jun 28;65(7):1213–1224. doi: 10.1016/0092-8674(91)90016-r. [DOI] [PubMed] [Google Scholar]
  8. Chenevert J., Corrado K., Bender A., Pringle J., Herskowitz I. A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains. Nature. 1992 Mar 5;356(6364):77–79. doi: 10.1038/356077a0. [DOI] [PubMed] [Google Scholar]
  9. Chenevert J., Valtz N., Herskowitz I. Identification of genes required for normal pheromone-induced cell polarization in Saccharomyces cerevisiae. Genetics. 1994 Apr;136(4):1287–1296. doi: 10.1093/genetics/136.4.1287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Choi K. Y., Satterberg B., Lyons D. M., Elion E. A. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell. 1994 Aug 12;78(3):499–512. doi: 10.1016/0092-8674(94)90427-8. [DOI] [PubMed] [Google Scholar]
  11. Clark K. L., Dignard D., Thomas D. Y., Whiteway M. Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating. Mol Cell Biol. 1993 Jan;13(1):1–8. doi: 10.1128/mcb.13.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cvrcková F., De Virgilio C., Manser E., Pringle J. R., Nasmyth K. Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev. 1995 Aug 1;9(15):1817–1830. doi: 10.1101/gad.9.15.1817. [DOI] [PubMed] [Google Scholar]
  13. Cvrcková F., Nasmyth K. Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation. EMBO J. 1993 Dec 15;12(13):5277–5286. doi: 10.1002/j.1460-2075.1993.tb06223.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hagen D. C., McCaffrey G., Sprague G. F., Jr Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1418–1422. doi: 10.1073/pnas.83.5.1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  16. Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ireland L. S., Johnston G. C., Drebot M. A., Dhillon N., DeMaggio A. J., Hoekstra M. F., Singer R. A. A member of a novel family of yeast 'zn-finger' proteins mediates the transition from stationary phase to cell proliferation. EMBO J. 1994 Aug 15;13(16):3812–3821. doi: 10.1002/j.1460-2075.1994.tb06692.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kidd S. Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins. Cell. 1992 Nov 13;71(4):623–635. doi: 10.1016/0092-8674(92)90596-5. [DOI] [PubMed] [Google Scholar]
  19. Koshland D., Kent J. C., Hartwell L. H. Genetic analysis of the mitotic transmission of minichromosomes. Cell. 1985 Feb;40(2):393–403. doi: 10.1016/0092-8674(85)90153-9. [DOI] [PubMed] [Google Scholar]
  20. Leberer E., Dignard D., Harcus D., Thomas D. Y., Whiteway M. The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components. EMBO J. 1992 Dec;11(13):4815–4824. doi: 10.1002/j.1460-2075.1992.tb05587.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liu H., Styles C. A., Fink G. R. Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science. 1993 Dec 10;262(5140):1741–1744. doi: 10.1126/science.8259520. [DOI] [PubMed] [Google Scholar]
  22. Lux S. E., John K. M., Bennett V. Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins. Nature. 1990 Mar 1;344(6261):36–42. doi: 10.1038/344036a0. [DOI] [PubMed] [Google Scholar]
  23. Ma H., Kunes S., Schatz P. J., Botstein D. Plasmid construction by homologous recombination in yeast. Gene. 1987;58(2-3):201–216. doi: 10.1016/0378-1119(87)90376-3. [DOI] [PubMed] [Google Scholar]
  24. Michaely P., Bennett V. The ANK repeat: a ubiquitous motif involved in macromolecular recognition. Trends Cell Biol. 1992 May;2(5):127–129. doi: 10.1016/0962-8924(92)90084-z. [DOI] [PubMed] [Google Scholar]
  25. Miyajima I., Nakafuku M., Nakayama N., Brenner C., Miyajima A., Kaibuchi K., Arai K., Kaziro Y., Matsumoto K. GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction. Cell. 1987 Sep 25;50(7):1011–1019. doi: 10.1016/0092-8674(87)90167-x. [DOI] [PubMed] [Google Scholar]
  26. Peterson J., Zheng Y., Bender L., Myers A., Cerione R., Bender A. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast. J Cell Biol. 1994 Dec;127(5):1395–1406. doi: 10.1083/jcb.127.5.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
  28. Printen J. A., Sprague G. F., Jr Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics. 1994 Nov;138(3):609–619. doi: 10.1093/genetics/138.3.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ramer S. W., Davis R. W. A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):452–456. doi: 10.1073/pnas.90.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  31. Schneider K. R., Smith R. L., O'Shea E. K. Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science. 1994 Oct 7;266(5182):122–126. doi: 10.1126/science.7939631. [DOI] [PubMed] [Google Scholar]
  32. Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993 Dec 16;366(6456):704–707. doi: 10.1038/366704a0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Simon M. N., De Virgilio C., Souza B., Pringle J. R., Abo A., Reed S. I. Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway. Nature. 1995 Aug 24;376(6542):702–705. doi: 10.1038/376702a0. [DOI] [PubMed] [Google Scholar]
  35. Stevenson B. J., Rhodes N., Errede B., Sprague G. F., Jr Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 1992 Jul;6(7):1293–1304. doi: 10.1101/gad.6.7.1293. [DOI] [PubMed] [Google Scholar]
  36. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  37. Wharton K. A., Johansen K. M., Xu T., Artavanis-Tsakonas S. Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell. 1985 Dec;43(3 Pt 2):567–581. doi: 10.1016/0092-8674(85)90229-6. [DOI] [PubMed] [Google Scholar]
  38. Whiteway M. S., Wu C., Leeuw T., Clark K., Fourest-Lieuvin A., Thomas D. Y., Leberer E. Association of the yeast pheromone response G protein beta gamma subunits with the MAP kinase scaffold Ste5p. Science. 1995 Sep 15;269(5230):1572–1575. doi: 10.1126/science.7667635. [DOI] [PubMed] [Google Scholar]
  39. Whiteway M., Hougan L., Dignard D., Thomas D. Y., Bell L., Saari G. C., Grant F. J., O'Hara P., MacKay V. L. The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein. Cell. 1989 Feb 10;56(3):467–477. doi: 10.1016/0092-8674(89)90249-3. [DOI] [PubMed] [Google Scholar]
  40. Wu C., Whiteway M., Thomas D. Y., Leberer E. Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae. J Biol Chem. 1995 Jul 7;270(27):15984–15992. doi: 10.1074/jbc.270.27.15984. [DOI] [PubMed] [Google Scholar]
  41. Zhao Z. S., Leung T., Manser E., Lim L. Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24. Mol Cell Biol. 1995 Oct;15(10):5246–5257. doi: 10.1128/mcb.15.10.5246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zheng Y., Cerione R., Bender A. Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. J Biol Chem. 1994 Jan 28;269(4):2369–2372. [PubMed] [Google Scholar]

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