scholarly journals Determinants of substrate specificity and biochemical properties of thesn-glycerol-3-phosphate ATP binding cassette transporter (UgpB-AEC2) ofEscherichia coli

2012 ◽  
Vol 86 (4) ◽  
pp. 908-920 ◽  
Author(s):  
Steven Wuttge ◽  
Martin Bommer ◽  
Franziska Jäger ◽  
Berta M. Martins ◽  
Sophie Jacob ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Biochemical Properties ◽  
Ofescherichia Coli ◽  
Atp Binding ◽  
Atp Binding Cassette Transporter ◽  
Atp Binding Cassette

scholarly journals Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter

1998 ◽  
Vol 9 (2) ◽  
pp. 523-543 ◽  
Author(s):  
Ralf Egner ◽  
Friederike E. Rosenthal ◽  
Anastasia Kralli ◽  
Dominique Sanglard ◽  
Karl Kuchler
Keyword(s):  
Drug Resistance ◽  
Substrate Specificity ◽  
Drug Transport ◽  
Transmembrane Domain ◽  
Substrate Recognition ◽  
Yeast Cells ◽  
Atp Binding ◽  
In Vitro Mutagenesis ◽  
Atp Binding Cassette Transporter ◽  
Atp Binding Cassette

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.

scholarly journals A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport

2003 ◽  
Vol 373 (3) ◽  
pp. 767-774 ◽  
Author(s):  
Hideyuki MITOMO ◽  
Ryo KATO ◽  
Akiko ITO ◽  
Shiho KASAMATSU ◽  
Yoji IKEGAMI ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Atp Binding ◽  
Transport Activity ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Atp Binding Cassette Transporter ◽  
293 Cells ◽  
Atp Binding Cassette

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg482, Gly482, and Thr482 variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg482]ABCG2 transports [3H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly482 and Thr482). Transport of methotrexate by [Arg482]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S-octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg482]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg482 is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

Sign in / Sign up

Export Citation Format

Share Document