AMINO-TERMINAL ASSEMBLY OF HUMAN P-GLYCOPROTEIN AT THE ENDOPLASMIC-RETICULUM IS DIRECTED BY COOPERATIVE ACTIONS OF 2 INTERNAL SEQUENCES

Transmembrane topology of polytopic integral membrane proteins is esta blished during protein synthesis at the endoplasmic reticulum membrane . For some polytopic proteins, sequential and independent signal, stop transfer, and/or signal anchor sequences contained in the nascent cha in direct this process. Here we define the topology of human P-glycopr otein (MDR1) through the first two transmembrane regions (TM1 and TM2, respectively) of the amino-terminal half of the protein. We show that unlike TM7 and TM8, which comprise homologous regions in the carboxyl half of the protein (Skach, W., Calayag, M. C., and Lingappa, V. (199 3) J. Biol. Chem. 268, 6903-6908), TM1 and TM2 achieve the orientation predicted by conventional structural models. However, TM1 and TM2 app ear to utilize a mechanism of biogenesis different in a key respect fr om that observed in multispanning proteins studied previously. TM1 and TM2, with their flanking regions, independently direct the topology o bserved for each of these sequences in the native protein. Each can in teract with signal recognition particle to direct targetting to the en doplasmic reticulum, nascent chain translocation, and correct transmem brane orientation. Unlike the transmembrane regions of previously stud ied multispanning membrane proteins, neither TM1 nor TM2 alone is suff icient to integrate the chain into the membrane. However, when TM1 and TM2 are both present, as occurs in native MDR1, integration is achiev ed. These results suggest that cooperative interactions between TM1 an d TM2 are necessary for chain integration and thus add a new complexit y to the current view of polytopic integral membrane protein biogenesi s.