Transmembrane Orientation and Topogenesis of the Third and Fourth Membrane-spanning Regions of Human P-Glycoprotein (MDR1)

Understanding how the multidrug resistance phenotype is manifest in human cancer cells will require insight into the mechanism of assembly, transmembrane topology, and intracellular trafficking of human P-glycoprotein (MDR1). Previously, we showed that MDR1 amino terminus biogenesis occurred through an unexpected interaction between novel topogenic sequence subtypes and that transmembrane topology of corresponding amino and carboxy halves was not equivalent We now investigate topology and topogenic activities of the third and fourth transmembrane regions (TM3 and TM4) of human MDR1 using protease protection of defined reporter epitopes expressed in Xenopus laevis oocytes. As was previously observed for TM1 and TM2, determinants in TM3 and TM4 exhibited cooperativity in directing proper assembly and transmembrane orientation. The signal sequence encompassing TM3 required residues from TM4 to reinitiate translocation of the MDR1 chain into the endoplasmic reticulum (ER) lumen. Remaining residues from TM4 terminated translocation and established a polytopic transmembrane topology in which TM3 and TM4 both spanned the membrane in the orientation predicted by hydropathy-based models. Remarkably, when translocating sequentially into the ER lumen, neither TM4 alone nor TM4 together with TM3 efficiently terminated translocation. Thus, MDR1 biogenesis required both the presence of these sequences and their proper orientation with respect to the ER translocation apparatus. This conclusion was supported by experiments in which TM3 and TM4 topology was reproduced in a defined chimeric protein which mimicked native MDR1 presentation. These additional variations on simple themes of protein topogenesis utilized by MDR1 demonstrate that events of complex protein biogenesis may be dissected and studied using protein chimeras with defined translocation properties.