Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes

Guo, Zhong, Johnston, Wayne A., Kovtun, Oleksiy, Mureev, Sergey, Brocker, Cornelia, Ungermann, Christian and Alexandrov, Kirill (2013) Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes. PloS One, 8 12: e81534.1-e81534.8. doi:10.1371/journal.pone.0081534


Author Guo, Zhong
Johnston, Wayne A.
Kovtun, Oleksiy
Mureev, Sergey
Brocker, Cornelia
Ungermann, Christian
Alexandrov, Kirill
Title Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes
Journal name PloS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2013-12-02
Year available 2013
Sub-type Article (original research)
DOI 10.1371/journal.pone.0081534
Open Access Status DOI
Volume 8
Issue 12
Start page e81534.1
End page e81534.8
Total pages 9
Place of publication San Francisco, CA United States
Publisher Public Library of Science
Language eng
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.
Formatted 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.
Keyword Macromolecular protein
Cell free protein
Multisubunit complexes
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID DP1094080
569652
Institutional Status UQ

 
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Created: Tue, 10 Dec 2013, 20:40:31 EST by Susan Allen on behalf of Institute for Molecular Bioscience