title: Amino-terminal assembly of human P-glycoprotein at the endoplasmic reticulum is directed by cooperative actions of two internal sequences.
abstract: Transmembrane topology of polytopic integral membrane proteins is established 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 chain direct this process. Here we define the topology of human P-glycoprotein (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. (1993) J. Biol. Chem. 268, 6903-6908), TM1 and TM2 achieve the orientation predicted by conventional structural models. However, TM1 and TM2 appear to utilize a mechanism of biogenesis different in a key respect from that observed in multispanning proteins studied previously. TM1 and TM2, with their flanking regions, independently direct the topology observed for each of these sequences in the native protein. Each can interact with signal recognition particle to direct targetting to the endoplasmic reticulum, nascent chain translocation, and correct transmembrane orientation. Unlike the transmembrane regions of previously studied multispanning membrane proteins, neither TM1 nor TM2 alone is sufficient to integrate the chain into the membrane. However, when TM1 and TM2 are both present, as occurs in native MDR1, integration is achieved. These results suggest that cooperative interactions between TM1 and TM2 are necessary for chain integration and thus add a new complexity to the current view of polytopic integral membrane protein biogenesis.
authors.affiliation: Department of Medicine, University of California, San Francisco 94143.
authors.forename: W R
authors.initials: WR
authors.lastname: Skach
authors.forename: V R
authors.initials: VR
authors.lastname: Lingappa
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System; D017931:DNA Primers / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins