title: Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions.
abstract: COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.
authors.affiliation: Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
authors.forename: Monika C
authors.initials: MC
authors.lastname: Sahlmüller
authors.forename: Jeroen R P M
authors.initials: JR
authors.lastname: Strating
authors.forename: Rainer
authors.initials: R
authors.lastname: Beck
authors.forename: Priska
authors.initials: P
authors.lastname: Eckert
authors.forename: Vincent
authors.initials: V
authors.lastname: Popoff
authors.forename: Mathias
authors.initials: M
authors.lastname: Haag
authors.forename: Andrea
authors.initials: A
authors.lastname: Hellwig
authors.forename: Imre
authors.initials: I
authors.lastname: Berger
authors.forename: Britta
authors.initials: B
authors.lastname: Brügger
authors.forename: Felix T
authors.initials: FT
authors.lastname: Wieland
mesh_terms: D000818:Animals; D001692:Biological Transport; D022181:COP-Coated Vesicles / Q000378:metabolism; D002460:Cell Line; D020756:Coatomer Protein / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D051379:Mice; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D020756:Coatomer Protein; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins