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- Title
Mapping protein-protein interactions of yeast xenobiotic efflux pump Pdr5p using the Integrated Membrane Yeast-Two Hybrid System: Pdr5p-Erg4p interaction important for Pdr5p function.
- Authors
Yang, Min-Han Michael; Shevelev, Igor; Stagljar, Igor
- Abstract
Background: Pdr5p is an ABC transporter protein in the pleiotropic drug resistance (PDR) family in Saccharomyces cerevisiae, which is homologous to the human P-glycoprotein important in cancer multi-drug resistance. Proteins like P-glycoprotein rarely exert their function alone, instead they are in constant dynamic interaction with other proteins and complexes that can modulate and influence their activity. However, it is difficult to study protein-protein interactions in human cells in a high- throughput manner. Using yeast as a model organism, this investigation aims to identify as many protein interactors of Pdr5p as possible using the Integrated Split-Ubiquitin Membrane-Yeast-Two Hybrid (iMYTH) System. Identifying Pdr5p's interactors in yeast will give clues as how to manage chemoresitance in cancers due to P-glycoprotein in human. Methods: Pdr5p's ORF is endogenously tagged with Cub-TF by homologous recombination. Sequencing and genetic testing were performed to confirm the PDR5 gene was inserted in-frame. The tagged cell were then subjected to a large scale transformation using plasmids containing yeast genomic library with NubG fused to the N-terminal side and plated onto -WAH plates. The clones were then subjected to plasmid isolation, and then transformed into MC1061 bacterial cells to obtain sufficient plasmid for sequencing. The sequence results were ran through BLAST on the "Saccharomyces Genome Database" to identify the prey proteins. False positive interactors were identified by performing a Bait- Dependency test by increasing concentrations of 3-amino-1,2,4 triazole. In the Bait Dependency test, the prey containing plasmids were transformed into the Pdr5p-Cub-TF, Ste24p-Cub-TF and Ycf1p-Cub- TF yeast strains. Lastly, drug sensitivity tests were performed on knock-out mutants of potential interactors of Pdr5p by plating on cycloheximide containing plates. Results: The iMYTH system yielded 18 unique protein interactors of Pdr5p of which 12 were confirmed using the Bait Dependency Test. Among the 12 true interactors of Pdr5p, Erg4p was confirmed to be in the same functional pathway as Pdr5p by using the cycloheximide drug sensitivity test. Conclusion: 12 true protein interactors of Pdr5p were identified of which Erg4p was shown to be important to the xenobiotic efflux pump activity of Pdr5p.
- Subjects
PROTEIN-protein interactions; XENOBIOTICS; SACCHAROMYCES cerevisiae; GLYCOPROTEINS; DRUG resistance; P-glycoprotein
- Publication
UBC Medical Journal, 2011, Vol 2, Issue 2, p30
- ISSN
1920-7425
- Publication type
Abstract