title: Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence.
abstract: P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Kast
authors.forename: V
authors.initials: V
authors.lastname: Canfield
authors.forename: R
authors.initials: R
authors.lastname: Levenson
authors.forename: P
authors.initials: P
authors.lastname: Gros
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D000939:Epitopes / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006388:Hemagglutinins; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016324:Sequence Tagged Sites; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary; D000939:Epitopes; D006388:Hemagglutinins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins