Drug‐Induced Permeabilization of S. cerevisiae

Maria D. Mayan1

1 Cell Cycle Group, MRC Clinical Sciences Centre, Imperial College, London, United Kingdom
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.2B
DOI:  10.1002/0471142727.mb1302bs92
Online Posting Date:  October, 2010
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Abstract

Budding yeast are not permeable to many drugs. This unit provides a protocol in which polygodial is used to permeabilize the cell membrane, thereby allowing budding yeast cells to be treated with drugs that otherwise would be ineffective. Curr. Protoc. Mol. Biol. 92:13.2B.1‐13.2B.4. © 2010 by John Wiley & Sons, Inc.

Keywords: permeabilization; Alpha‐amantin; DRB; S. cerevisiae

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • S. cerevisiae growing on a plate
  • Yeast growth medium (see recipe)
  • 1.25 mg/ml polygodial in absolute ethanol (see recipe)
  • Drug for introduction into yeast cells—e.g., α‐amanitin (AM; Sigma) or 5,6‐dichlorobenzimidazole 1‐β‐D‐ribofuranoside (DRB; Sigma; see Table 13.2.1).
  • Dimethyl sulfoxide (DMSO)
  • 100‐ and 500‐ml glass flasks, sterile
NOTE: In general, perform all incubations at 30°C (except when using thermosensitive strains) since polygodial works well between 23° to 39°C.
Table 3.2.1   MaterialsEffectiveness of Polygodial on Different Drugs in Yeast

Drug Tested concentrations Effect of the treatment using 0.39 µg/ml of polygodial
Alpha‐amanitin 10 µg/ml 1 hr; 20 µg/ml 1 hr; 50 µg/ml 1 hr and 8 hr Decreased 80% to 90% of RNAP‐II transcription a (M. Mayan, unpub. observ.); 50 µg/ml over 8 hr also affects RNAP‐I transcription a (M. Mayan, unpub. observ.)
DRB 25 µg/ml 4 hr; 50 µg/ml 40 min, 1 hr, and 4 hr; 100 µg/ml 30 min, 1 hr, and 4 hr 25 and 50 µg/ml for 40 min decreases 70% to 80% of RNAP‐II transcripts; higher concentrations eliminate nearly all studied RNAP‐II primary transcripts (∼90%) a (M. Mayan, unpub. observ.)
Actinomycin D 10 µg/ml 2 hr; 12 µg/ml 48 hr See Taniguchi et al. ( )
Rifampicin 100 µg/ml 2 hr and 48 hr; See Taniguchi et al. ( )
Cyclohexamine 25 µg/ml 1 hr; 50 µg/ml 1 hr; 100 µg/ml 30 min 25 and 50 µg/ml of cyclohexamine decreases 80% of protein detected by immunoblot (β‐actin); 100 µg/ml decreases 90% of β‐actin levels after 30 min (M. Mayan, unpub. observ.)

 aPrimary RNA levels were quantified, using SYBr green real‐time PCR.
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Figures

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Literature Cited

Literature Cited
   Andres, M.–I., Forsby, A.F., and Walum, E. 1997. Polygodial‐induced noradrenaline release in human neuroblastoma SH‐SY5Y cells. Toxicol. In Vitro 11:509‐511.
   Battaner, E. and Kumar, B.V. 1974. Rifampin: Inhibition of ribonucleic acid synthesis after potentiation by amphotericin B in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 5:371‐376.
   Bushnell, D.A., Cramer, P., and Kornberg, R.D. 2002. Structural basis of transcription: Alpha‐amanitin‐RNA polymerase II cocrystal at 2.8 A resolution. Proc. Natl. Acad. Sci. U.S.A. 99:1218‐1222.
   Elion, E.A. and Warner, J.R. 1986. An RNA polymerase I enhancer in Saccharomyces cerevisiae. Mol. Cell. Biol. 6:2089‐2097.
   Kaur, R. and Bachhawat, A.K. 1999. The yeast multidrug resistance pump, Pdr5p, confers reduced drug resistance in erg mutants of Saccharomyces cerevisiae. Microbiology 145:809‐818.
   Kubo, I. and Taniguchi, M. 1988. Polygodial, an antifungal potentiator. J. Nat. Prod. 51:22‐29.
   Kwan, C.N., Medoff, G., Kobayashi, G.S., Schlessinger, D., and Raskas, H.J. 1972. Potentiation of the antifungal effects of antibiotics by amphotericin B. Antimicrob. Agents Chemother. 2:61‐65.
   Lee, D.H. and Goldberg, A.L. 1996. Selective inhibitors of the proteasome‐dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae. J. Biol. Chem. 271:27280‐27284.
   Medoff, G., Kobayashi, G.S., Kwan, C.N., Schlessinger, D., and Venkov, P. 1972. Potentiation of rifampicin and 5‐fluorocytosine as antifungal antibiotics by amphotericin B (yeast‐membrane permeability‐ribosomal RNA‐eukaryotic cell‐synergism. Proc. Natl. Acad. Sci. U.S.A. 69:196‐199.
   Mittleman, B., Zandomeni, R., and Weinmann, R. 1983. Mechanism of action of 5,6‐dichloro‐1‐beta‐D‐ribofuranosylbenzimidazole. II. A resistant human cell mutant with an altered transcriptional machinery. J. Mol. Biol. 165:461‐473.
   Nitiss, J. and Wang, J.C. 1988. DNA topoisomerase‐targeting antitumor drugs can be studied in yeast. Proc. Natl. Acad. Sci. U.S.A. 85:7501‐7505.
   Schwartz, S.N., Medoff, G., Kobayashi, G.S., Kwan, C.N., and Schlessinger, D. 1972. Antifungal properties of polymyxin B and its potentiation of tetracycline as an antifungal agent. Antimicrob. Agents Chemother. 2:36‐40.
   Somesh, B.P., Reid, J., Liu, W.F., Søgaard, T.M., Erdjument‐Bromage, H., Tempst, P., and Svejstrup, J.Q. 2005. Multiple mechanisms confining RNA polymerase II ubiquitylation to polymerases undergoing transcriptional arrest. Cell 121:913‐923.
   Taniguchi, M., Yano, Y., Motoba, K., Tanaka, T., Oi, S., Haraguchi, H., Hashimoto, K., and Kubo, I. 1988. Polygodial‐induced sensitivity to rifampicin and actinomycin D of Saccharomyces cerevisiae (biological chemistry). Agric. Biol. Chem. 52:1881‐1883.
   Vennström, B., Persson, H., Pettersson, U., and Philipson, L. 1979. A DRB (5,6 dichloro‐beta‐D‐ribofuranosylbenzimidazole)‐resistant adenovirus mRNA. Nucleic Acids Res. 7:1405‐1418.
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