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P-glycoprotein (Pgp) is a polytopic membrane protein responsible for multidrug resistance in cancer cells. Previously, we have used a coupled cell-free translation/translocation system to investigate the membrane orientation of Pgp sequences and have made the unexpected observation that predicted transmembrane (TM) segments from both the NH₂-terminal and COOH-terminal halves inserted in microsomal membranes in two different orientations (Zhang, J.-T., Duthie, M., and Ling, V.(1993) J. Biol. Chem. 268, 15101-15110). How these topological forms of Pgp are regulated is not known. In the present study, we have used site-directed mutagenesis to investigate if the amino acids surrounding the internal TM segments of Pgp may affect their orientation. We discovered that the charged amino acids flanking TM4 are important in determining the membrane orientation of the NH₂-terminal half molecule of Pgp …