title: Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes.
abstract: Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.
authors: Department of Cell and Molecular Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors: Zhong
authors: Z
authors: Guo
authors: Wayne
authors: W
authors: Johnston
authors: Oleksiy
authors: O
authors: Kovtun
authors: Sergey
authors: S
authors: Mureev
authors: Cornelia
authors: C
authors: Bröcker
authors: Christian
authors: C
authors: Ungermann
authors: Kirill
authors: K
authors: Alexandrov
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D007891:Leishmania / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D021122:Protein Subunits