TRANSMEMBRANE ORGANIZATION OF MOUSE P-GLYCOPROTEIN DETERMINED BY EPITOPE INSERTION AND IMMUNOFLUORESCENCE

P-glycoprotein (P-gp) is an integral membrane protein that causes mult idrug resistance when overexpressed in tumor cells. Efforts to identif y the position and polarity of its 12 putative transmembrane (TM) doma ins have so far failed to yield a consistent topological model. Recent ly, we have described a method for topology mapping based on the inser tion of a small antigenic peptide epitope (YPYDVPDYA) in predicted int ra- or extracellular loops of the protein. The tagged proteins are the n functionally expressed in Chinese hamster ovary cells, and the polar ity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously loc alized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellula r (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemis try 34, 4402-4411). We have now inserted single epitope tags at positi ons 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, 8 49, 887, 961, and 1024 resulted in functional proteins, whereas insert ions at positions 207, 276, 782, 797, and 815 abrogated the capacity o f P-gp to confer multidrug resistance. The epitope tags inserted at po sitions 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-TM1 1, 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 do main topology with intracellular amino and carboxyl termini and ATP bi nding sites and an extracellular glycosylated loop (TM1-TM2) in agreem ent with hydropathy prediction. These results are clearly distinct fro m those obtained by the analysis of truncated P-gps in vitro and in he terologous expression systems.