title: Organization of the Sec61 translocon, studied by high resolution native electrophoresis.
abstract: Cotranslational translocation of polypeptides into the ER is controlled by the dynamic interaction of ribosome and translocon components. Analysis of the steps involved in this process by high resolution techniques such as gel electrophoresis is precluded by the high molecular masses of these complexes. We show, here, that modifications to standard native electrophoresis protocols can overcome these problems and lead to an increase in mass range and resolution. Using the modified technique, we show that ER ribosome anchored membrane protein (RAMP) complexes resolve into 3 stable and semistable complexes which range in size between 4 and 8 MDa and are sensitive to relevant concentrations of divalent metals. We demonstrate the molecular composition of the complexes and identify a number of modular components that differentiate them. The components that are common to all three RAMP complexes include the OST translocon subcomplex, Glucosidase I and microtubule tethering protein CLIMP63. The two larger complexes further include the kinesin motor binding protein p180 and Sec61, and the largest complex includes the TRAP translocon component and apoptotic regulator BAP31. On the lumenal side, the BiP cochaperone ERdj3 resides with the three RAMP complexes. Our observations may hint at how subcompartmentalization is achieved in the ER membrane continuum.
authors.affiliation: Department of Biochemistry, and McGill Cancer Centre, McIntyre Medical Building, McGill University, 3655 Promenade Sir William Osler, Montreal, Canada. kurt.dejgaard@mcgill.ca
authors.forename: Kurt
authors.initials: K
authors.lastname: Dejgaard
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Theberge
authors.forename: Hannah
authors.initials: H
authors.lastname: Heath-Engel
authors.forename: Eric
authors.initials: E
authors.lastname: Chevet
authors.forename: Michel L
authors.initials: ML
authors.lastname: Tremblay
authors.forename: David Y
authors.initials: DY
authors.lastname: Thomas
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D010449:Peptide Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods; D012270:Ribosomes / Q000378:metabolism; D000069816:SEC Translocation Channels
chemical_list: D008565:Membrane Proteins; D000069816:SEC Translocation Channels