Membrane Topology of a Cysteine-less Mutant of Human P-glycoprotein

doi:10.1074/jbc.270.2.843

Membrane Topology of a Cysteine-less Mutant of Human P-glycoprotein (*)

Tip W. Loo and
David M. Clarke(§)

From the Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada

^§ Scholar of the Medical Research Council of Canada. To whom correspondence and reprint requests should be addressed:
Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
Tel.: 416-978-1105; Fax: 416-978-8765.

Abstract

A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect to its ability to confer resistance to various drugs. The cysteine-less P-glycoprotein-A52 retained the ability to confer resistance to vinblastine, colchicine, doxorubicin, and actinomycin D with only a small decrease in efficiency relative to wild-type enzyme. Cysteine residues were then reintroduced into predicted extracellular or cytoplasmic loops of the cysteine-less P-glycoprotein-A52, and the topology of the protein was determined using membrane-permeant and impermeant thiol-specific reagents. It was found that 8 of 15 cysteine residues introduced into P-glycoprotein-A52 could be biotinylated, when cells expressing the mutant P-glycoprotein were incubated with membrane-permeant biotin maleimide. Biotinylation of a cysteine residue placed in predicted extracellular loops between transmembrane segment (TM) 5 and TM6, TM7 and TM8, or TM11 and TM12 was blocked by pretreatment of the cells with a membrane-impermeant maleimide, suggesting that these residues have an extracellular location. By contrast, biotinylation of cysteine residues located in the predicted cytoplasmic loops between TM2 and TM3, TM4 and TM5, TM8 and TM9, or TM10 and TM11 were not blocked by pretreatment with membrane impermeant maleimide, suggesting that these residues were in the cytoplasm. These results are consistent with the model of P-glycoprotein, which predicts six transmembrane segments in each of the two homologous halves of the molecule.

Footnotes

↵* This research was supported by a grant (to D. M. C.) as part of a group grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MDR

multidrug resistance

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

TM

transmembrane segment

biotin maleimide

3-(N-maleimidylpropionyl)biocytin

stilbenedisulfonate maleimide

4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid

SERCA

sarcoendoplasmic reticulum Ca-ATPase.
↵²D. M. Clarke and T. W. Loo, unpublished observations.
- Received August 1, 1994.
- Revision received October 27, 1994.