Solubilization and characterization of the overexpressed PDR5 multidrug resistance nucleotide triphosphatase of yeast
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Updated: Oct 23, 2026
High-throughput Yeast Plasmid Overexpression Screen
Published on: July 27, 2011
A Decottignies1, M Kolaczkowski, E Balzi
1Unité de Biochimie Physiologique, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Abstract:
A 160-kDa plasma membrane protein of the yeast Saccharomyces cerevisiae was overexpressed by mutating the PDR1 or the PDR3 transcription factor gene. The protein is the membrane-bound ATP binding cassette transporter PDR5 (Balzi, E., Wang, M., Leterme, S., Van Dyck, L., and Goffeau, A. (1994) J. Biol. Chem. 269, 2206-2214). PDR5 was solubilized with n-dodecyl-beta-D-malto-side and separated from the PMA1 plasma membrane H(+)-ATPase by glycerol gradient centrifugation. The PDR5 protein hydrolyzes nucleoside diphosphates and triphosphates. This activity is sensitive to low concentrations of vanadate, of oligomycin, and of a variety of hydrophobic compounds. Many of these properties liken PDR5 to the purified mammalian P-glycoprotein responsible for multidrug resistance.
Insights
Researchers identified and characterized the PDR5 protein, an ATP binding cassette transporter in yeast. This transporter
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- The plasma membrane of Saccharomyces cerevisiae contains various transporters crucial for cellular function.
- Understanding these transporters aids in deciphering cellular mechanisms, including drug resistance.
- The PDR1 and PDR3 transcription factors regulate the expression of several genes in yeast.
Purpose of the Study:
- To identify and characterize a 160-kDa plasma membrane protein in Saccharomyces cerevisiae.
- To investigate the function and properties of the PDR5 protein, an ATP binding cassette transporter.
- To compare the characteristics of PDR5 with mammalian P-glycoprotein involved in multidrug resistance.
Main Methods:
- Overexpression of the PDR5 protein by mutating PDR1 or PDR3 transcription factor genes.
- Solubilization of PDR5 using n-dodecyl-beta-D-malto-side.
- Separation of PDR5 from PMA1 H(+)-ATPase via glycerol gradient centrifugation.
- Enzymatic assays to determine nucleoside phosphate hydrolysis activity.
- Sensitivity analysis using vanadate, oligomycin, and hydrophobic compounds.
Main Results:
- The 160-kDa plasma membrane protein was identified as the ATP binding cassette transporter PDR5.
- PDR5 was successfully solubilized and separated from the PMA1 H(+)-ATPase.
- PDR5 exhibits nucleoside diphosphate and triphosphate hydrolyzing activity.
- This activity is inhibited by vanadate, oligomycin, and hydrophobic compounds.
Conclusions:
- The PDR5 transporter in Saccharomyces cerevisiae shares functional similarities with mammalian P-glycoprotein.
- These similarities suggest a conserved mechanism for transport and potential roles in resistance.
- Further research into PDR5 can provide insights into multidrug resistance mechanisms.
