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
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
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 15:40
Last modification date
20/08/2019 13:25