Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter

Details

Serval ID
serval:BIB_040973231336
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter
Journal
Molecular Biology of the Cell
Author(s)
Egner  R., Rosenthal  F. E., Kralli  A., Sanglard  D., Kuchler  K.
ISSN
1059-1524 (Print)
Publication state
Published
Issued date
02/1998
Volume
9
Number
2
Pages
523-43
Notes
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: Feb
Abstract
Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.
Keywords
ATP-Binding Cassette Transporters/antagonists & inhibitors/chemistry/*genetics Amino Acid Sequence Amino Acid Substitution Antibiotics, Antifungal/pharmacology Biological Transport Carrier Proteins/genetics/physiology Cell Membrane/chemistry Cloning, Molecular Cycloheximide/pharmacology DNA-Binding Proteins/genetics/physiology Dexamethasone/metabolism/pharmacology Drug Resistance, Microbial/*genetics Drug Resistance, Multiple/*genetics Estradiol/metabolism Gene Expression/drug effects Heat-Shock Proteins/genetics/physiology Membrane Proteins/antagonists & inhibitors/chemistry/*genetics Molecular Sequence Data Mutagenesis Rhodamine 123 Rhodamines/metabolism/pharmacology Saccharomyces cerevisiae/*drug effects/genetics *Saccharomyces cerevisiae Proteins Sequence Alignment Substrate Specificity Tacrolimus/*pharmacology Tacrolimus Binding Proteins
Pubmed
Web of science
Create date
25/01/2008 14:40
Last modification date
20/08/2019 12:25
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