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. 1996 Nov 1;135(3):741–751. doi: 10.1083/jcb.135.3.741

Role of the ABC transporter Ste6 in cell fusion during yeast conjugation

PMCID: PMC2121058  PMID: 8909547

Abstract

Though early stages of yeast conjugation are well-mimicked by treatment with pheromones, the final degradation of the cell wall and membrane fusion of mating that leads to cytoplasmic mixing may require separate signals. Mutations that blocked cell fusion during mating in Saccharomyces cerevisiae were identified in a multipartite screen. The three tightest mutations proved to be partial-function alleles of the ABC-transporter gene STE6 required for transport of a-factor. The ste6(cefl-1) allele was recovered and sequenced. The ste6(cefl-1) allele contained a stop codon predicted to truncate Ste6 at amino acid residue 862 (of 1290). The ste6(cef) mutations reduced, but did not eliminate, expression of a-factor. Light and electron microscopy revealed that unlike ste6 null mutations which block mating before the formation of mating pairs, the ste6(cef) (cell fusion) alleles permitted early steps in mating to proceed normally but blocked at a late stage in conjugation where mating partners were encased by a single cell wall and separated by only a thin layer of cell wall material we term the fusion wall. Morphologically the prezygotes appeared symmetrical with successful cell wall fusion at the periphery of the region of cell contact. Responses to a-factor were efficiently induced in partner cells under mating conditions as expected given the symmetric appearance of the prezygotes. A strain expressing a ste6(K1093A) mutation that conferred export of a twofold to fourfold higher level of a-factor than ste6(cef) did not accumulate prezygotes during mating which could indicate a tight threshold of a-factor signaling required for mating. However, mating to an sst2 partner which has a greatly increased sensitivity to a-factor did not suppress the fusion defect of a ste6(cef) strain. Overexpression of the structural gene for a-factor also did not suppress the fusion defect. It is possible that a-factor or STE6 play more complex roles in cell fusion.

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Selected References

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  1. Berkower C., Michaelis S. Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO J. 1991 Dec;10(12):3777–3785. doi: 10.1002/j.1460-2075.1991.tb04947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Betz R., Duntze W. Purification and partial characterization of a factor, a mating hormone produced by mating-type-a cells from Saccharomyces cerevisiae. Eur J Biochem. 1979 Apr;95(3):469–475. doi: 10.1111/j.1432-1033.1979.tb12986.x. [DOI] [PubMed] [Google Scholar]
  3. Byers B., Goetsch L. Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J Bacteriol. 1975 Oct;124(1):511–523. doi: 10.1128/jb.124.1.511-523.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CONTI S. F., BROCK T. D. ELECTRON MICROSCOPY OF CELL FUSION IN CONJUGATING HANSENULA WINGEI. J Bacteriol. 1965 Aug;90:524–533. doi: 10.1128/jb.90.2.524-533.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chan R. K., Melnick L. M., Blair L. C., Thorner J. Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae. J Bacteriol. 1983 Aug;155(2):903–906. doi: 10.1128/jb.155.2.903-906.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chan R. K., Otte C. A. Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones. Mol Cell Biol. 1982 Jan;2(1):11–20. doi: 10.1128/mcb.2.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dietzel C., Kurjan J. Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone. Mol Cell Biol. 1987 Dec;7(12):4169–4177. doi: 10.1128/mcb.7.12.4169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eliasson L., Renström E., Ammälä C., Berggren P. O., Bertorello A. M., Bokvist K., Chibalin A., Deeney J. T., Flatt P. R., Gäbel J. PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells. Science. 1996 Feb 9;271(5250):813–815. doi: 10.1126/science.271.5250.813. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Elion E. A., Trueheart J., Fink G. R. Fus2 localizes near the site of cell fusion and is required for both cell fusion and nuclear alignment during zygote formation. J Cell Biol. 1995 Sep;130(6):1283–1296. doi: 10.1083/jcb.130.6.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ellis R. E., Jacobson D. M., Horvitz H. R. Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans. Genetics. 1991 Sep;129(1):79–94. doi: 10.1093/genetics/129.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fearon K., McClendon V., Bonetti B., Bedwell D. M. Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. J Biol Chem. 1994 Jul 8;269(27):17802–17808. [PubMed] [Google Scholar]
  13. Fenno J. C., Shaikh A., Spatafora G., Fives-Taylor P. The fimA locus of Streptococcus parasanguis encodes an ATP-binding membrane transport system. Mol Microbiol. 1995 Mar;15(5):849–863. doi: 10.1111/j.1365-2958.1995.tb02355.x. [DOI] [PubMed] [Google Scholar]
  14. Higgins C. F. The ABC of channel regulation. Cell. 1995 Sep 8;82(5):693–696. doi: 10.1016/0092-8674(95)90465-4. [DOI] [PubMed] [Google Scholar]
  15. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  16. Hyde S. C., Emsley P., Hartshorn M. J., Mimmack M. M., Gileadi U., Pearce S. R., Gallagher M. P., Gill D. R., Hubbard R. E., Higgins C. F. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature. 1990 Jul 26;346(6282):362–365. doi: 10.1038/346362a0. [DOI] [PubMed] [Google Scholar]
  17. Jackson C. L., Hartwell L. H. Courtship in S. cerevisiae: both cell types choose mating partners by responding to the strongest pheromone signal. Cell. 1990 Nov 30;63(5):1039–1051. doi: 10.1016/0092-8674(90)90507-b. [DOI] [PubMed] [Google Scholar]
  18. Kuchler K., Sterne R. E., Thorner J. Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells. EMBO J. 1989 Dec 20;8(13):3973–3984. doi: 10.1002/j.1460-2075.1989.tb08580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kurihara L. J., Beh C. T., Latterich M., Schekman R., Rose M. D. Nuclear congression and membrane fusion: two distinct events in the yeast karyogamy pathway. J Cell Biol. 1994 Aug;126(4):911–923. doi: 10.1083/jcb.126.4.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lipke P. N., Taylor A., Ballou C. E. Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells. J Bacteriol. 1976 Jul;127(1):610–618. doi: 10.1128/jb.127.1.610-618.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lipke P. N., Wojciechowicz D., Kurjan J. AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating. Mol Cell Biol. 1989 Aug;9(8):3155–3165. doi: 10.1128/mcb.9.8.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luciani M. F., Chimini G. The ATP binding cassette transporter ABC1, is required for the engulfment of corpses generated by apoptotic cell death. EMBO J. 1996 Jan 15;15(2):226–235. [PMC free article] [PubMed] [Google Scholar]
  23. Manavalan P., Smith A. E., McPherson J. M. Sequence and structural homology among membrane-associated domains of CFTR and certain transporter proteins. J Protein Chem. 1993 Jun;12(3):279–290. doi: 10.1007/BF01028190. [DOI] [PubMed] [Google Scholar]
  24. Marcus S., Caldwell G. A., Miller D., Xue C. B., Naider F., Becker J. M. Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone. Mol Cell Biol. 1991 Jul;11(7):3603–3612. doi: 10.1128/mcb.11.7.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marsh L., Herskowitz I. STE2 protein of Saccharomyces kluyveri is a member of the rhodopsin/beta-adrenergic receptor family and is responsible for recognition of the peptide ligand alpha factor. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3855–3859. doi: 10.1073/pnas.85.11.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marsh L., Neiman A. M., Herskowitz I. Signal transduction during pheromone response in yeast. Annu Rev Cell Biol. 1991;7:699–728. doi: 10.1146/annurev.cb.07.110191.003411. [DOI] [PubMed] [Google Scholar]
  27. Marsh L. Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist. Mol Cell Biol. 1992 Sep;12(9):3959–3966. doi: 10.1128/mcb.12.9.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McCaffrey G., Clay F. J., Kelsay K., Sprague G. F., Jr Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987 Aug;7(8):2680–2690. doi: 10.1128/mcb.7.8.2680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McGrath J. P., Varshavsky A. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature. 1989 Aug 3;340(6232):400–404. doi: 10.1038/340400a0. [DOI] [PubMed] [Google Scholar]
  30. Michaelis S., Herskowitz I. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol Cell Biol. 1988 Mar;8(3):1309–1318. doi: 10.1128/mcb.8.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Michaelis S. STE6, the yeast a-factor transporter. Semin Cell Biol. 1993 Feb;4(1):17–27. doi: 10.1006/scel.1993.1003. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  34. Segall J. E. Polarization of yeast cells in spatial gradients of alpha mating factor. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8332–8336. doi: 10.1073/pnas.90.18.8332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sen M., Marsh L. Noncontiguous domains of the alpha-factor receptor of yeasts confer ligand specificity. J Biol Chem. 1994 Jan 14;269(2):968–973. [PubMed] [Google Scholar]
  36. Sprague G. F., Jr Assay of yeast mating reaction. Methods Enzymol. 1991;194:77–93. doi: 10.1016/0076-6879(91)94008-z. [DOI] [PubMed] [Google Scholar]
  37. Strazdis J. R., MacKay V. L. Reproducible and rapid methods for the isolation and assay of a-factor, a yeast mating hormone. J Bacteriol. 1982 Sep;151(3):1153–1161. doi: 10.1128/jb.151.3.1153-1161.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tomeo M. E., Fenner G., Tove S. R., Parks L. W. Effect of sterol alterations on conjugation in Saccharomyces cerevisiae. Yeast. 1992 Dec;8(12):1015–1024. doi: 10.1002/yea.320081204. [DOI] [PubMed] [Google Scholar]
  39. Trueheart J., Boeke J. D., Fink G. R. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol Cell Biol. 1987 Jul;7(7):2316–2328. doi: 10.1128/mcb.7.7.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wilson K. L., Herskowitz I. Negative regulation of STE6 gene expression by the alpha 2 product of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Nov;4(11):2420–2427. doi: 10.1128/mcb.4.11.2420. [DOI] [PMC free article] [PubMed] [Google Scholar]

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