Deletion and insertion mutants of the multidrug transporter.
The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs. To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations. The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain. However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance. An internal deletion of the amino-terminal half, which removed residues 140-229, is also nonfunctional. Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance. Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity. These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter.
Currier
S J
SJ
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Ueda
K
K
Willingham
M C
MC
Pastan
I
I
Gottesman
M M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D002872:Chromosome Deletion; D003078:Colchicine / Q000494:pharmacology; D004251:DNA Transposable Elements; D004351:Drug Resistance; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D009154:Mutation; D010641:Phenotype
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D008562:Membrane Glycoproteins; D003078:Colchicine