Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

Autor(en): Guo, Zhong
Johnston, Wayne
Kovtun, Oleksiy
Mureev, Sergey
Broecker, Cornelia
Ungermann, Christian 
Alexandrov, Kirill
Stichwörter: ARCHITECTURE; EXPRESSION; FUSION; INTERACTS; Multidisciplinary Sciences; NUCLEOTIDE EXCHANGE; PURIFICATION; Science & Technology - Other Topics; SYSTEM; YPT1P
Erscheinungsdatum: 2013
Herausgeber: PUBLIC LIBRARY SCIENCE
Journal: PLOS ONE
Volumen: 8
Ausgabe: 12
Zusammenfassung: 
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.
ISSN: 19326203
DOI: 10.1371/journal.pone.0081534

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