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Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1992 Feb;3(2):167–180. doi: 10.1091/mbc.3.2.167

Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.

M Kawamukai 1, J Gerst 1, J Field 1, M Riggs 1, L Rodgers 1, M Wigler 1, D Young 1
PMCID: PMC275516  PMID: 1550959

Abstract

We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP.

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Selected References

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  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brutlag D. L., Dautricourt J. P., Maulik S., Relph J. Improved sensitivity of biological sequence database searches. Comput Appl Biosci. 1990 Jul;6(3):237–245. doi: 10.1093/bioinformatics/6.3.237. [DOI] [PubMed] [Google Scholar]
  3. Colicelli J., Birchmeier C., Michaeli T., O'Neill K., Riggs M., Wigler M. Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase. Proc Natl Acad Sci U S A. 1989 May;86(10):3599–3603. doi: 10.1073/pnas.86.10.3599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colicelli J., Field J., Ballester R., Chester N., Young D., Wigler M. Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase. Mol Cell Biol. 1990 Jun;10(6):2539–2543. doi: 10.1128/mcb.10.6.2539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeFeo-Jones D., Scolnick E. M., Koller R., Dhar R. ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature. 1983 Dec 15;306(5944):707–709. doi: 10.1038/306707a0. [DOI] [PubMed] [Google Scholar]
  6. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Field J., Vojtek A., Ballester R., Bolger G., Colicelli J., Ferguson K., Gerst J., Kataoka T., Michaeli T., Powers S. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990 Apr 20;61(2):319–327. doi: 10.1016/0092-8674(90)90812-s. [DOI] [PubMed] [Google Scholar]
  8. Field J., Vojtek A., Ballester R., Bolger G., Colicelli J., Ferguson K., Gerst J., Kataoka T., Michaeli T., Powers S. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990 Apr 20;61(2):319–327. doi: 10.1016/0092-8674(90)90812-s. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Gerst J. E., Ferguson K., Vojtek A., Wigler M., Field J. CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex. Mol Cell Biol. 1991 Mar;11(3):1248–1257. doi: 10.1128/mcb.11.3.1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoffman C. S., Winston F. Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes Dev. 1991 Apr;5(4):561–571. doi: 10.1101/gad.5.4.561. [DOI] [PubMed] [Google Scholar]
  12. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kataoka T., Broek D., Wigler M. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell. 1985 Dec;43(2 Pt 1):493–505. doi: 10.1016/0092-8674(85)90179-5. [DOI] [PubMed] [Google Scholar]
  14. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M. Genetic analysis of yeast RAS1 and RAS2 genes. Cell. 1984 Jun;37(2):437–445. doi: 10.1016/0092-8674(84)90374-x. [DOI] [PubMed] [Google Scholar]
  15. Kawamukai M., Ferguson K., Wigler M., Young D. Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe. Cell Regul. 1991 Feb;2(2):155–164. doi: 10.1091/mbc.2.2.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matsumoto K., Uno I., Oshima Y., Ishikawa T. Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2355–2359. doi: 10.1073/pnas.79.7.2355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McLeod M., Beach D. Homology between the ran1+ gene of fission yeast and protein kinases. EMBO J. 1986 Dec 20;5(13):3665–3671. doi: 10.1002/j.1460-2075.1986.tb04697.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Nikawa J., Sass P., Wigler M. Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Oct;7(10):3629–3636. doi: 10.1128/mcb.7.10.3629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pelham H. R. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:1–23. doi: 10.1146/annurev.cb.05.110189.000245. [DOI] [PubMed] [Google Scholar]
  21. Powers S., Kataoka T., Fasano O., Goldfarb M., Strathern J., Broach J., Wigler M. Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell. 1984 Mar;36(3):607–612. doi: 10.1016/0092-8674(84)90340-4. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Sass P., Field J., Nikawa J., Toda T., Wigler M. Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9303–9307. doi: 10.1073/pnas.83.24.9303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Suzuki N., Choe H. R., Nishida Y., Yamawaki-Kataoka Y., Ohnishi S., Tamaoki T., Kataoka T. Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8711–8715. doi: 10.1073/pnas.87.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Toda T., Cameron S., Sass P., Wigler M. SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits. Genes Dev. 1988 May;2(5):517–527. doi: 10.1101/gad.2.5.517. [DOI] [PubMed] [Google Scholar]
  27. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  28. Vojtek A., Haarer B., Field J., Gerst J., Pollard T. D., Brown S., Wigler M. Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae. Cell. 1991 Aug 9;66(3):497–505. doi: 10.1016/0092-8674(81)90013-1. [DOI] [PubMed] [Google Scholar]
  29. Wigler M., Field J., Powers S., Broek D., Toda T., Cameron S., Nikawa J., Michaeli T., Colicelli J., Ferguson K. Studies of RAS function in the yeast Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):649–655. doi: 10.1101/sqb.1988.053.01.074. [DOI] [PubMed] [Google Scholar]
  30. Xu H. P., Wang Y., Riggs M., Rodgers L., Wigler M. Biological activity of the mammalian RAP genes in yeast. Cell Regul. 1990 Sep;1(10):763–769. doi: 10.1091/mbc.1.10.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yamawaki-Kataoka Y., Tamaoki T., Choe H. R., Tanaka H., Kataoka T. Adenylate cyclases in yeast: a comparison of the genes from Schizosaccharomyces pombe and Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5693–5697. doi: 10.1073/pnas.86.15.5693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]

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