Membrane orientation of transmembrane segments 11 and 12 of MDR- and non-MDR-associated P-glycoproteins.

P-glycoprotein (Pgp) is a polytopic plasma membrane protein thought to function as a drug efflux pump. Two functional groups of Pgp have been identified in mammalian cells. One group (classes I and II) is associated with MDR and the other (class III) is not. Transmembrane (TM) sequences in Pgp have been postulated to be important for determining drug specificity. TM11 and TM12 have been predicted to bind drugs and play an important role in determining drug specificity of MDR-associated Pgps. Whether or not the membrane insertion and orientation of these TM segments differ amongst the different classes of Pgp has not been examined directly. In this study, we showed that membrane insertion and orientation of TM11 and TM12 of the MDR-associated Pgp may differ from the non-MDR-associated Pgp using an in vitro transcription and translation system. Charged amino acids surrounding TM domains are thought to be important in determining the topology of membrane proteins. The positively charged amino acids surrounding TM11 and TM12 of these two forms of Pgp are different. By site-directed mutagenesis we showed that these amino acids may affect the membrane orientation but not membrane insertion of these TMs. These results raise the possibility that a difference in membrane anchorage may be a underlying cause for the functional difference between the two groups of Pgp.